FGF-21 conjugate preparation

Abstract

The present application provides pharmaceutical formulations comprising PEGylated FGF-21, e.g., an FGF-21 conjugate, and one or more stabilizers, such as the chelating agent DPTA. The formulations may be further stabilized by incorporating a surfactant, such as polysorbate 80, and / or adjusting the pH to about 7.1. Methods of manufacture, methods of treatment, and kits are also provided.

Description

[Technical field]

[0001] References to sequence listings submitted electronically via EFS-WEB The contents of the Sequence Listing (Name: 3338_208PC03_SeqListing.txt; Size: 11,049 bytes; and Creation Date: January 5, 2021), submitted electronically with this application, are hereby incorporated by reference in their entirety. Field

[0002] The present application relates, inter alia, to formulations comprising an FGF-21 polypeptide, e.g., a PEGylated FGF-21 polypeptide (PEG-FGF-21), stabilized using a chelating agent (e.g., DTPA), a surfactant (e.g., PS80), and having a specific pH range (e.g., about 7.1), for administration via various routes, including, for example, subcutaneous administration. [Background technology]

[0003] Fibroblast growth factors (FGFs) are polypeptides that are widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3:239-243, 1991) that play crucial roles in multiple physiological functions (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998;Burgess, WH et al., Annu. Rev. Biochem. 58:575-606 (1989)). According to the literature, the FGF family consists of at least 22 members (Reuss et al., Cell Tissue Res. 313:139-157 (2003)).

[0004] FGF-21 can be used for the treatment of diseases and conditions associated with fibrosis. Fibrosis is the formation of excess fibrous connective tissue in organs or tissues. Excessive deposition of fibrous tissue is associated with pathological conditions that can lead to organ or tissue dysfunction. Affected organs can include the lung (pulmonary or pulmonary fibrosis), liver (liver or liver fibrosis), kidney (renal or kidney fibrosis), and heart (cardiac fibrosis). Fibrosis can also affect other tissues and organs, including joints, skin, intestine, bone marrow, and others. Exemplary fibrotic conditions or diseases include, but are not limited to, non-alcoholic steatohepatitis (NASH), which affects the liver; diabetic kidney disease and diabetic nephropathy, which affect the kidney; and metabolic heart failure, which affects the heart. For example, NASH is characterized by fat accumulation, inflammation, and damage in the liver of people who consume little or no alcohol, and the disease can lead to cirrhosis of the liver. NASH tends to be diagnosed in middle-aged people who are overweight or obese, who have elevated blood levels of certain lipids and who often have diabetes or prediabetes.

[0005] FGF-21 has been reported to undergo proteolysis in vivo, form aggregates in vitro, and be prone to deamidation (Gimeno and Moller, Trends Endocrinol Metab. 2014 June; 25(6):303-11; U.S. Patent No. 8,361,963; Hecht et al., PLoS One. 2012; 7(11):e49345; U.S. Patent Publication No. 2007 / 0293430; WO2006 / 0065582), potentially limiting the shelf-life of pharmaceutical compositions containing FGF-21. Aggregates and deamidated forms of therapeutic polypeptides can potentially increase immunogenicity (see US Department of Health and Human Services, "Immunogenicity Assessment for Therapeutic Protein Products," August 2014; Subramanyam (ed.), "Therapeutic Protein Immunogenicity Focus Group Newsletter," American Association of Pharmaceutical Scientists, Vol. 1, Issue 3 (December 2011)). Summary of the Invention [Problem to be solved by the invention]

[0006] The present disclosure addresses, inter alia, the need for pharmaceutical formulations that include FGF-21 polypeptides, and in particular that address problems associated with making pharmaceutical formulations that include mutant FGF-21 polypeptides. [Means for solving the problem]

[0007] The present disclosure relates to a pharmaceutical formulation comprising: (i) a fibroblast growth factor 21 (FGF-21) polypeptide conjugated to a polyethylene glycol (PEG) moiety ("FGF-21 conjugate"), and (ii) an aminopolycarboxylic acid cation chelator, wherein the formulation has improved stability compared to a reference formulation that does not contain the aminopolycarboxylic acid cation chelator, and wherein the FGF-21 conjugate has a structure represented by Formula I: [ka] wherein n is any integer.

[0008] In some embodiments, the PEG moiety is conjugated to an unnatural amino acid in the FGF-21 polypeptide. In some embodiments, the unnatural amino acid in the FGF-21 polypeptide is a phenylalanine derivative. In some embodiments, the phenylalanine derivative is para-acetyl-L-phenylalanine.

[0009] In some embodiments, the FGF-21 polypeptide comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3, where the polypeptide has FGF-21 activity. In some embodiments, the unnatural amino acid is at amino acid residue 109, which corresponds to SEQ ID NO:3. In some embodiments, the FGF-21 polypeptide comprises the sequence set forth in SEQ ID NO:1. In some embodiments, the FGF-21 conjugate corresponds to the compound of SEQ ID NO:2.

[0010] In some embodiments, n in formula I is about 500 to about 900 ethylene glycol units, about 600 to about 800 ethylene glycol units, about 650 to about 750 ethylene glycol units, or about 670 to about 690 ethylene glycol units. In some embodiments, n is between about 670 and about 690 ethylene glycol units, e.g., about 681 ethylene glycol units.

[0011] In some embodiments, the FGF-21 conjugate corresponds to the compound of SEQ ID NO: 4. In some embodiments, the FGF-21 conjugate is in the L conformation. In some embodiments, the FGF-21 conjugate is in the D conformation. In some embodiments, the FGF-21 conjugate is present at a concentration between about 1 mg / ml and about 40 mg / ml. In some embodiments, the FGF-21 conjugate is present at a concentration of about 10 mg / ml or about 20 mg / ml. In some embodiments, the FGF-21 conjugate is present at a concentration of about 20 mg / ml.

[0012] In some embodiments, the FGF-21 conjugate is present in an amount between about 1 mg and about 40 mg per unit dose. In some embodiments, the FGF-21 conjugate is present in an amount of about 1 mg per unit dose, about 5 mg per unit dose, about 10 mg per unit dose, about 20 mg per unit dose, or about 40 mg per unit dose. In some embodiments, the FGF-21 conjugate is present in an amount of about 10 mg or about 20 mg per unit dose.

[0013] In some aspects, the pharmaceutical formulation comprises: (a) The decrease in the deamidation rate of the polypeptide relative to a reference formulation when stored at 40° C. for approximately one month; (b) a reduction in the rate of aggregation of high molecular weight (HMW) polypeptides when stored at 40° C. for about one month relative to a reference formulation; or (c) Both (a) and (b) The patient may exhibit one or more of the following symptoms:

[0014] In some embodiments, the aminopolycarboxylic acid cation chelator prevents or mitigates oxidation of one or more methionines. In some embodiments, the methionines correspond to methionine 1 (Met1) and / or methionine 169 (Met169) of the FGF-21 polypeptide. In some embodiments, oxidation of the methionines is prevented or mitigated by the chelator at 25° C. and / or 40° C. In some embodiments, the aminopolycarboxylic acid cation chelator is diethylenetriaminepentaacetic acid (DTPA). In some embodiments, the DTPA cation chelator is present in an amount of between about 10 μM and about 100 μM DTPA, between about 20 μM and about 90 μM DTPA, between about 30 μM and about 80 μM DTPA, between about 25 μM and about 75 μM DTPA, or between about 40 μM and about 60 μM DTPA, or between about 30 μM and about 70 μM DTPA, or between about 40 μM and about 70 μM DTPA. In some embodiments, the DTPA cation chelator is present in an amount of about 40 μM, about 45 μM, about 50 μM, about 55 μM, or about 60 μM DTPA.

[0015] In some embodiments, the pH of the formulation is greater than 6.5, greater than 6.6, greater than 6.7, greater than 6.8, greater than 6.9, greater than 7.0, greater than 7.1, greater than 7.2, greater than 7.3, greater than 7.4, or greater than 7.5. In some embodiments, the pH is between about 6.7 to about 7.5, about 6.8 to about 7.5, about 6.9 to about 7.4, about 7.0 to about 7.3, about 7.1 to 7.2, about 7.1 to about 7.3, about 7.1 to about 7.4, or about 7.1 to about 7.5. In some embodiments, the pH is about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the pharmaceutical formulation is more stable than a reference formulation having a pH of 6.5.

[0016] In some embodiments, the pharmaceutical formulation further comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the non-anionic surfactant is a polysorbate. In some embodiments, the polysorbate is polyoxyethylene (20) sorbitan monooleate (polysorbate 80). In some embodiments, the surfactant that is polysorbate 80 is present in an amount of about 0.01% to about 0.1% (w / v), about 0.02% to about 0.09% (w / v), about 0.03% to about 0.08% (w / v), about 0.04% to about 0.07% (w / v), or about 0.05% to about 0.06% (w / v). In some embodiments, the surfactant that is polysorbate 80 is present in an amount of at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), or at least about 0.1% (w / v). In some embodiments, the surfactant mitigates the formation of particulates and / or bubbles when agitated on a shaker.

[0017] In some embodiments, the pharmaceutical formulation further comprises an amino acid buffer. In some embodiments, the amino acid buffer is histidine. In some embodiments, the histidine buffer is present in an amount of about 10 mM to about 100 mM histidine, about 20 mM to about 90 mM histidine, about 30 mM to about 80 mM histidine, about 40 mM to about 70 mM histidine, about 10 mM to about 30 mM histidine, about 15 mM to about 25 mM histidine, about 17.5 to about 22.5 mM histidine, or about 40 mM to about 60 mM histidine. In some embodiments, the histidine buffer is present in an amount of about 10 mM histidine, about 15 mM histidine, about 20 mM histidine, about 25 mM histidine, about 30 mM histidine, about 35 mM histidine, about 40 mM histidine, about 45 mM histidine, or about 50 mM histidine.

[0018] In some embodiments, the pharmaceutical formulation further comprises an osmotic modifier. In some embodiments, the osmotic modifier comprises a sugar. In some embodiments, the sugar is sucrose. In some embodiments, the osmotic modifier is sucrose in an amount of about 100 mM to about 1 M sucrose, about 200 mM to about 900 mM sucrose, about 300 mM to about 800 mM sucrose, about 400 mM to about 700 mM sucrose, or about 500 mM to about 600 mM sucrose. In some embodiments, the osmotic modifier that is sucrose is present in an amount of about 100 mM sucrose, about 200 mM sucrose, about 300 mM sucrose, about 400 mM sucrose, about 500 mM sucrose, about 600 mM sucrose, about 700 mM sucrose, about 800 mM sucrose, about 900 mM sucrose, or about 1 M sucrose.

[0019] In some aspects, the formulation is formulated for subcutaneous administration. In some aspects, the formulation is formulated for subcutaneous administration with a safety syringe. In some aspects, the formulation is formulated for daily or weekly administration. In some aspects, the formulation is an aqueous formulation.

[0020] The present disclosure relates to (i) FGF-21 conjugate; (ii) histidine at a concentration between about 10 mM and about 50 mM; (iii) sucrose at a concentration between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration between about 10 μM and about 100 μM A pharmaceutical formulation comprising: the pH of the formulation is between about 6.7 and about 7.5; The FGF-21 conjugate has formula I: [ka] Including, The present invention provides a pharmaceutical formulation, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0021] The present disclosure also provides (i) FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 μM A pharmaceutical formulation comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a pharmaceutical formulation wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0022] Also, (i) FGF-21 conjugate; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 μM A pharmaceutical formulation comprising: The pH is 7.1, The FGF-21 conjugate has formula I: [ka] Including, Also provided is a pharmaceutical formulation wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0023] The present disclosure relates to (i) FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; and (iii) sucrose at a concentration of about 600 mM A pharmaceutical formulation comprising: The pH is about 7.0; The FGF-21 conjugate has formula I: [ka] Including, The present invention provides a pharmaceutical formulation, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0024] The present disclosure relates to (i) FGF-21 conjugate; (ii) histidine at a concentration of 20 mM; and (iii) sucrose at a concentration of 600 mM A pharmaceutical formulation comprising: The pH is 7.0, The FGF-21 conjugate has formula I: [ka] Including, The present invention provides a pharmaceutical formulation, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0025] The present disclosure also provides (i) an FGF-21 conjugate at a concentration of about 10 mg / mL; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM A pharmaceutical formulation comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a pharmaceutical formulation wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0026] The present disclosure also provides (i) an FGF-21 conjugate at a concentration of about 20 mg / mL; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM A pharmaceutical formulation comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a pharmaceutical formulation wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0027] The present disclosure relates to (i) FGF-21 conjugate at a concentration of 10 mg / mL; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 uM A pharmaceutical formulation comprising: The pH is 7.0, The FGF-21 conjugate has formula I: [ka] Including, The present invention provides a pharmaceutical formulation, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0028] The present disclosure relates to (i) PEG-FGF21 of SEQ ID NO: 2 or 4 at a concentration of 20 mg / mL; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 uM A pharmaceutical formulation comprising: The pH is 7.0, The FGF-21 conjugate has formula I: [ka] Including, The present invention provides a pharmaceutical formulation, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0029] The present disclosure relates to a method for improving the stability of a pharmaceutical formulation comprising a fibroblast growth factor 21 (FGF-21) polypeptide conjugated to a polyethylene glycol (PEG) moiety ("FGF-21 conjugate"), the method comprising the step of admixing an aminopolycarboxylic acid cation chelator, the formulation having improved stability compared to a reference formulation not containing the aminopolycarboxylic acid cation chelator, the FGF-21 conjugate having a structure represented by formula I: [ka] Including, The method includes the steps of: n is between about 670 and about 690, for example, about 681; and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0030] In some embodiments of the methods disclosed herein, the PEG moiety is conjugated to a non-natural amino acid in the FGF-21 polypeptide. In some embodiments, the non-natural amino acid in the FGF-21 polypeptide is para-acetyl-L-phenylalanine. In some embodiments, the FGF-21 polypeptide is an FGF-21 polypeptide of SEQ ID NO: 1. In some embodiments, the FGF-21 conjugate is in an L conformation. In some embodiments, the FGF-21 conjugate is in a D conformation. In some embodiments, the improved stability includes (i) an increase in the physical stability of the polypeptide, (ii) an increase in the chemical stability of the polypeptide, or (iii) both (i) and (ii). In some embodiments, the increased physical stability includes (i) prevention or reduction of polypeptide aggregation, (ii) prevention or reduction of polypeptide fragmentation, or (iii) both (i) and (ii). In some embodiments, the increased chemical stability includes (i) preventing or reducing deamidation of the polypeptide, (ii) preventing or reducing oxidation of the polypeptide, or (iii) both (i) and (ii). In some embodiments, the improved stability includes one or more of: (a) reducing the deamidation rate of the polypeptide when stored at 40°C for about one month relative to a reference formulation; (b) reducing the aggregation rate of high molecular weight (HMW) polypeptide when stored at 40°C for about one month relative to a reference formulation; or (c) both (a) and (b). In some embodiments, the improved stability includes preventing or mitigating oxidation of one or more methionines. In some embodiments, the methionines correspond to Met1 and / or Met169 of the FGF-21 polypeptide. In some embodiments, the oxidation of methionines is prevented or mitigated at 25°C and / or 40°C.

[0031] In some embodiments of the methods disclosed herein, the aminopolycarboxylic acid cation chelator is DTPA. In some embodiments, the DTPA cation chelator is present in an amount of between about 10 μM and about 100 μM DTPA, between about 20 μM and about 90 μM DTPA, between about 25 μM and about 75 μM DTPA, between about 40 μM and about 60 μM DTPA, between about 30 μM and about 70 μM DTPA, between about 30 μM and about 80 μM DTPA, or between about 40 μM and about 70 μM DTPA. In some embodiments, the DTPA cation chelator is present in an amount of about 40 μM, about 45 μM, about 50 μM, about 55 μM, or about 60 μM DTPA.

[0032] In some embodiments of the methods disclosed herein, the method further comprises adjusting the pH to greater than 6.5, greater than 6.6, greater than 6.7, greater than 6.8, greater than 6.9, or greater than 7.0. In some embodiments, the pH is adjusted to between about 6.8 and about 7.5, or between about 6.9 and about 7.4, or between about 7.0 and about 7.3, or between about 7.1 and 7.2, or between about 7.1 and about 7.3, or between about 7.1 and about 7.4, or between about 7.1 and about 7.5. In some embodiments, the adjusted pH is about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the formulation is more stable than a reference formulation having a pH of 6.5.

[0033] In some embodiments of the methods disclosed herein, the method further comprises mixing a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the non-anionic surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the surfactant, which is polysorbate 80, is mixed in an amount of about 0.01% to about 0.1% (w / v), about 0.02% to about 0.09% (w / v), about 0.03% to about 0.08% (w / v), about 0.04% to about 0.07% (w / v), or about 0.05% to about 0.06% (w / v). In some embodiments, the surfactant, polysorbate 80, is mixed in an amount of at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), or at least about 0.1% (w / v). In some embodiments, the surfactant mitigates particulate formation and / or air bubble formation when agitated on a shaker.

[0034] In some embodiments of the methods disclosed herein, the method further comprises mixing an amino acid buffer. In some embodiments, the amino acid buffer is histidine. In some embodiments, the histidine buffer is mixed in an amount of about 10 mM to about 100 mM histidine, about 20 mM to about 90 mM histidine, about 30 mM to about 80 mM histidine, about 40 mM to about 70 mM histidine, about 10 mM to about 30 mM histidine, about 15 mM to about 25 mM histidine, about 17.5 mM to about 22.5 mM histidine, or about 40 mM to about 60 mM histidine. In some embodiments, the histidine buffer is mixed in an amount of about 10 mM histidine, about 15 mM histidine, about 20 mM histidine, about 25 mM histidine, about 30 mM histidine, about 35 mM histidine, about 40 mM histidine, about 45 mM histidine, or about 50 mM histidine.

[0035] In some embodiments of the methods disclosed herein, the method further comprises mixing an osmotic pressure adjusting agent. In some embodiments, the osmotic pressure adjusting agent comprises a sugar. In some embodiments, the sugar is sucrose. In some embodiments, the osmotic pressure adjusting agent that is sucrose is mixed in an amount of about 100 mM to about 1 M sucrose, about 200 mM to about 900 mM sucrose, about 300 mM to about 800 mM sucrose, about 400 mM to about 700 mM sucrose, or about 500 mM to about 600 mM sucrose. In some embodiments, the osmotic modifier that is sucrose is mixed in an amount of about 100 mM sucrose, about 200 mM sucrose, about 300 mM sucrose, about 400 mM sucrose, about 500 mM sucrose, about 600 mM sucrose, about 700 mM sucrose, about 800 mM sucrose, about 900 mM sucrose, or about 1 M sucrose.

[0036] In some aspects, the formulation is an aqueous formulation. In some aspects, the disclosure provides pharmaceutical formulations prepared according to the methods disclosed herein.

[0037] The present disclosure also provides (i) FGF-21 conjugate; (ii) histidine at a concentration between about 10 mM and about 50 mM; (iii) sucrose at a concentration between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration between about 10 μM and about 100 μM A vial comprising: the pH of the formulation is between about 6.7 and about 7.5; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a vial, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0038] The present disclosure also provides (i) about 5 mg to about 20 mg of FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; and (iii) sucrose at a concentration of about 600 mM A vial comprising: The pH is about 7.0; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a vial, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0039] The present disclosure also provides (i) about 10 mg to about 20 mg of FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM A vial comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a vial, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0040] The present disclosure also provides (i) about 10 mg of FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM A vial comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a vial, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0041] The present disclosure also provides (i) about 20 mg of FGF-21 conjugate; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM A vial comprising: The pH is about 7.1; The FGF-21 conjugate has formula I: [ka] Including, Also provided is a vial, wherein n is between about 670 and about 690, for example, about 681, and the FGF-21 polypeptide comprises SEQ ID NO:1.

[0042] The present disclosure also provides a kit or article of manufacture comprising (i) a pharmaceutical formulation disclosed herein, or a vial disclosed herein, and (ii) instructions for use.

[0043] The present disclosure also provides a method for treating or preventing a disease or condition associated with fibrosis and / or diabetes in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical formulation disclosed herein, the vial disclosed herein, or the kit disclosed herein. In some embodiments, the disease or condition is diabetes. In some embodiments, the diabetes is type 2 diabetes. In some embodiments, the disease or condition is non-alcoholic steatohepatitis (NASH). In some embodiments, administering to the subject an effective amount of the pharmaceutical formulation reduces liver stiffness, reduces body fat percentage, reduces body weight, reduces liver weight to body weight ratio, reduces liver lipid content, reduces liver fibrosis area, reduces fasting blood glucose level, reduces fasting triglyceride level, reduces LDL cholesterol level, reduces ApoB level, reduces ApoC level, increases HDL cholesterol, or any combination thereof. In some embodiments, the FGF-21 conjugate is administered in a flat dose of about 20 mg. In some embodiments, the FGF-21 conjugate is administered at a one week dosing interval. In some embodiments, the pharmaceutical formulation is administered subcutaneously. In some embodiments, the pharmaceutical formulation is administered subcutaneously using a safety syringe. In some embodiments, administration of the pharmaceutical formulation to a subject results in a decrease in the level of FGF-21 in comparison to an untreated subject or to a level in the subject prior to administration of the pharmaceutical formulation. (i) reducing liver fat levels; (ii) reducing the level of liver damage; (iii) reduced levels of fibrosis; (iv) decreased serum Pro-C3 (N-terminal type III collagen propeptide) levels, a biomarker of fibrosis; (v) decreased alanine aminotransferase (ALT) levels; (vi) decreased aspartate aminotransferase (AST) levels; (vii) elevated serum adiponectin levels; (viii) reduction in plasma LDL levels; (ix) increased plasma HDL levels; (x) reduction in plasma triglyceride levels; (xi) reducing liver stiffness levels; or (xii) any combination of these This results in:

[0044] In some embodiments, the FGF-21 polypeptide may be conjugated to a PEG that is approximately 30 kDa. [Brief description of the drawings]

[0045] [Figure 1] Figures 1A and 1B show the deamidation of PEG-FGF-21 as a function of formulation composition. Figure 1A shows the deamidation in a formulation without pentetic acid (DTPA) or polysorbate 80. Figure 1B shows the deamidation in a formulation with pentetic acid (DTPA) and polysorbate 80.

[0046] [Diagram 2] 2A and 2B show the metal-catalyzed oxidation of PEG-FGF-21 polypeptide at methionine 1 (FIG. 2A) and methionine 169 (FIG. 2B) in the presence and absence of 50 μM DTPA (also known as pentetic acid).

[0047] [Diagram 3] Figures 3A and 3B show the effect of pH on the aggregation of PEG-FGF-21. Figure 3A shows the aggregation as a function of time, pH, and buffer system used. Figure 3B shows the aggregation at different temperatures and times (5°C, 14 months; 25°C, 1 month; 40°C, 1 day) and different pHs.

[0048] [Figure 4] Figures 4A and 4B show the aggregation of PEG-FGF-21 as a function of formulation composition. Figure 4A shows aggregation in a formulation without pentetic acid (DTPA) or polysorbate 80. Figure 4B shows aggregation in a formulation with pentetic acid (DTPA) and polysorbate 80.

[0049] [Diagram 5] Figure 5 shows that high concentrations of PEG-FGF-21 without PS80 became turbid after 24 h (left) or 6 h of wrist action shaking (right) on an orbital shaker at 300 rpm. Addition of between 0.01% and 0.1% (w / v) polysorbate 80 reduced sample turbidity.

[0050] [Figure 6] 6 shows a schematic representation of a PEGylated FGF-21 conjugate of the present disclosure; in particular, an example where the unnatural amino acid in the FGF-21 conjugate is para-acetyl-phenylalanine, e.g., para-acetyl-L-phenylalanine, where n represents the number of ethylene glycol units contained in the PEG polymer.

[0051] [Figure 7] FIG. 7 shows a schematic representation of a specific PEG linker containing 681 ethylene glycol units that can be fused or conjugated in a site-specific manner to an FGF-21 polypeptide (e.g., an FGF-21 polypeptide containing an unnatural amino acid such as para-acetyl-phenylalanine) to provide an FGF-21 conjugate of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present disclosure provides stabilized pharmaceutical formulations comprising fibroblast growth factor 21 (FGF-21) conjugates, such as, for example, PEG-FGF-21 (e.g., SEQ ID NO: 2 or 4). The presence of an aminopolycarboxylic acid cation chelator, such as DTPA, has been observed to mitigate oxidation of one or more amino acid residues, e.g., methionine, in the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4.

[0053] Incorporation of DTPA into a formulation containing the FGF-21 conjugate disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, also reduces the rate of deamidation of the FGF-21 polypeptide upon storage, and also reduces the rate of high molecular weight aggregation upon storage. Further stabilization can be achieved by adjusting the pH of the formulation to 7.1. In addition, the formulation can be further stabilized by adding a surfactant such as polysorbate 80.

[0054] The present disclosure also provides methods of manufacturing the disclosed formulations, as well as formulations made by applying the disclosed methods. Also provided are methods of treating or preventing diseases associated with fibrosis, such as NASH and diabetes, comprising administering the disclosed stabilized formulations to a subject in need thereof. definition

[0055] In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly set forth otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions shall be set forth throughout this application.

[0056] The present disclosure includes embodiments in which exactly one member of the group is present, employed, or otherwise relevant in a given product or process. The present disclosure includes embodiments in which more than one, or all, of the group members are present, employed, or otherwise relevant in a given product or process.

[0057] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "a," (or "an"), as well as "one or more" and "at least one," may be used interchangeably. In certain embodiments, the terms "a" or "an" mean "single." In other embodiments, the terms "a" or "an" include "two or more" or "multiple." Thus, for example, reference to an "FGF-21 conjugate" is a reference to one or more such proteins or conjugates, and includes equivalents thereof known to those of skill in the art, and so forth.

[0058] Furthermore, "and / or" as used herein shall be understood as a specific disclosure of each of the two specified features or components, with or without the other feature or component. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0059] As used herein, the term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within one standard deviation or more, as per the practice in the art. Alternatively, "about" may mean within a range of up to 20%. Furthermore, particularly when speaking of biological systems or processes, the term may mean within up to an order of magnitude, or up to 5-fold, of the value. When a particular value or composition is presented in this application and claims, unless otherwise stated, the meaning of "about" shall be assumed to be within an acceptable error range for the particular value or composition. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical values. Thus, "about 10 to 20" means "about 10 to about 20". In general, the term "about" can modify a numerical value above and below the stated value by a variation above or below (higher or lower), for example, of ten percent.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general definition of many of the terms used in this disclosure.

[0061] Wherever an embodiment is described herein using the term "comprising," it is understood that otherwise similar embodiments described using the terms "consisting of" and / or "consisting essentially of" are also provided.

[0062] Units, prefixes, and symbols are expressed in their International System of Units (SI) acceptable form. Numerical ranges are inclusive of the range-defined numbers. Unless otherwise indicated, amino acid sequences are written from left to right, in amino to carboxy orientation. The subheadings presented herein are not limitations of the various aspects of the disclosure that may be provided by reference to the specification as a whole. Thus, the terms defined immediately below are fully defined by reference to the specification as a whole.

[0063] Amino acids are referred to herein by their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0064] Aggregation: The term "aggregation" refers to the tendency of a polypeptide, such as the FGF-21 polypeptide portion of an FGF-21 conjugate disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, to form non-covalently linked complexes with other molecules, such as other molecules of the same polypeptide, thereby forming high molecular weight complexes. An exemplary method for measuring aggregate formation includes analytical size exclusion chromatography, as described in the Examples herein. The relative amount of aggregation can be determined, for example, with respect to a reference compound to identify a polypeptide that has reduced aggregation. The relative amount of aggregation can also be determined, for example, with respect to a reference formulation to identify a formulation in which an FGF-21 conjugate disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, has reduced aggregation.

[0065] Amino acid substitution: The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent or reference sequence (e.g., a wild-type sequence) with another amino acid residue. An amino acid may be substituted in a parent or reference sequence (e.g., a wild-type polypeptide sequence) for example, via chemical peptide synthesis or through recombinant methods known in the art. Thus, reference to a "substitution at position X" refers to the replacement of the amino acid present at position X with an alternative amino acid residue. In some embodiments, the substitution pattern may be described according to the scheme AnY, where A is the one-letter code corresponding to the naturally occurring or originally occurring amino acid at position n, and Y is the replacement amino acid residue. In other embodiments, the substitution pattern may be described according to the scheme An(YZ), where A is the one-letter code corresponding to the amino acid residue replacing the naturally occurring or originally occurring amino acid at position n, and Y and Z are the alternative replacement amino acid residues that may replace A.

[0066] In the context of the present disclosure, substitutions (even when referred to as amino acid substitutions) are made at the nucleic acid level, i.e., replacement of an amino acid residue by an alternative amino acid residue is made by replacing a codon encoding a first amino acid with a codon encoding a second amino acid.

[0067] About: As used herein, the term "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0068] Associated with: As used herein with respect to a disease, the term "associated with" means that the symptom, measurement, characteristic, or condition in question is associated with the diagnosis, onset, presence, or progression of the disease. An association may be causally associated with the disease, but need not be causally associated with the disease.

[0069] Biologically active: The term "biologically active" as applied to the molecules disclosed herein, e.g., the FGF-21 conjugates disclosed herein, e.g., the PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, means any substance that can affect any physical or biochemical property of a biological system, biological pathway, biological molecule, or biological interaction of an organism. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended to diagnose, cure, mitigation, treatment, or prevention of a disease or condition, e.g., a disease or condition associated with fibrosis in humans or other animals, or to otherwise enhance the physical or mental health of a human or animal.

[0070] Conservative amino acid substitution: "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, a family of amino acid residues with similar side chains is defined, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, when an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. In another embodiment, stretches of amino acids can be conservatively replaced with structurally similar stretches that differ in the order and / or composition of side chain family members.

[0071] Non-conservative amino acid substitutions include (i) substitutions in which a residue having an electropositive side chain (e.g., Arg, His, or Lys) is replaced with or by an electronegative residue (e.g., Glu or Asp); (ii) substitutions in which a hydrophilic residue (e.g., Ser or Thr) is replaced with or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) substitutions in which a cysteine ​​or proline is replaced with or by any other residue; or (iv) substitutions in which a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) is replaced with or by a residue having a small side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0072] Other amino acid substitutions can also be used. For example, for the amino acid alanine, the substitution can be any one of D-alanine, glycine, beta-alanine, L-cysteine, and D-cysteine. For lysine, the substitution can be any one of D-lysine, arginine, D-arginine, homoarginine, methionine, D-methionine, ornithine, or D-ornithine. In general, substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are: (i) polar residues, such as serine or threonine, are replaced with (or by) hydrophobic residues, such as leucine, isoleucine, phenylalanine, or alanine; (ii) cysteine ​​residues are replaced with (or by) any other residue; (iii) residues with electropositive side chains, such as lysine, arginine, or histidine, are replaced with (or by) residues with electronegative side chains, such as glutamic acid or aspartic acid; or (iv) residues with bulky side chains, such as phenylalanine, are replaced with (or by) residues without such side chains, such as glycine. The possibility that one of the above non-conservative substitutions may change the functional properties of a protein may also be correlated with the substitution position in the context of the functionally important regions of the protein: thus, some non-conservative substitutions may have little or no effect on biological properties.

[0073] Conserved: As used herein, the term "conserved" refers to amino acid residues of a polypeptide sequence that are amino acid residues that occur unchanged in the same position in two or more sequences that are being compared, respectively. Relatively conserved amino acids are those that are more closely conserved among the sequences than nucleotides or amino acids that appear elsewhere in the sequences.

[0074] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. Sequence conservation may apply to the entire length of a polynucleotide or polypeptide or to portions, regions, or features thereof.

[0075] Deamidation: The term "deamidation" refers to the tendency of amino acid residues in a polypeptide to spontaneously undergo deamidation reactions, thereby changing the chemical structure of the amino acid and potentially affecting the function of the polypeptide. Exemplary methods for measuring deamidation are disclosed in the Examples herein. The relative amount of deamidation can be determined, for example, against a reference compound to identify a polypeptide that has reduced deamidation. The relative amount of deamidation can also be determined against a reference formulation to identify a formulation in which the FGF-21 conjugates disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, have reduced deamidation.

[0076] Fibrosis-associated disease: The term "fibrosis-associated disease" includes diseases, disorders, and conditions in which fibrosis has been observed to occur or in which fibrosis is known or believed to be associated with or contribute to the pathogenesis, progression, or symptomology of the disease, or in which fibrosis is known or believed to occur as the disease progresses.

[0077] Fibrosis can affect organs or tissues such as the pancreas, lungs, heart, kidneys, liver, eyes, nervous system, bone marrow, lymph nodes, endocardium, or retroperitoneum. Exemplary diseases associated with fibrosis include nonalcoholic steatohepatitis (NASH), liver fibrosis, pre-cirrhosis, cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, pulmonary or pulmonary fibrosis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection site fibrosis, renal or kidney fibrosis, chronic kidney disease, diabetic kidney disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myofibrosis, heart failure, metabolic heart failure, cardiac fibrosis, cataract fibrosis, cataract, ocular scarring, pancreatic fibrosis, skin fibrosis, intestinal fibrosis, intestinal stenosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, Crohn's disease, retroperitoneal fibrosis, keloids, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, arthrofibrosis, pemphigoid fibrosis, psoriasis ... The diseases include, but are not limited to, erythropoietin syndrome, Dupuytren's contracture, diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, fatty liver, viral hepatitis, biliary disease, primary hemochromatosis, drug-related cirrhosis, idiopathic cirrhosis, Wilson's disease, and alpha-1 antitrypsin deficiency, interstitial lung disease (ILD), human fibrotic lung disease, macular degeneration, nephrogenic retinopathy, vitreoretinopathy, myocardial fibrosis, Graves' ophthalmopathy, drug-induced ergotine poisoning, cardiovascular disease, atherosclerosis / restenosis, hypertrophic scar, primary or idiopathic myofibrosis, and inflammatory bowel disease (including but not limited to collagenous colitis). In some embodiments, the disease associated with fibrosis may include liver fibrosis, renal or kidney fibrosis, pulmonary or lung fibrosis, and cardiac or heart fibrosis. In some embodiments, the disease associated with fibrosis may be liver fibrosis. In some aspects, the disease associated with fibrosis can be NASH.

[0078] Effective amount: As used herein for the formulations comprising the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, the term "effective amount" refers to an amount sufficient to produce beneficial or desired results, such as clinical results, and thus "effective amount" depends on the context in which it is applied.For example, in the context of administering the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 to treat NASH, an effective amount of the FGF-21 conjugate is, for example, an amount sufficient to improve liver fat, liver damage, or fibrosis (e.g., reducing liver fat, liver damage, or fibrosis in relation to the level in untreated subjects or in relation to the level in subjects before administering treatment). In some embodiments, the amount of FGF-21 polypeptide in the formulations disclosed herein is based on Slope Spectroscopy, e.g., as measured by an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0079] In some embodiments, an effective amount of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, for treating NASH may alter the level of one or more fibrosis biomarkers relative to the level in an untreated subject or relative to the level in the subject prior to administering the treatment: for example, may lower serum Pro-C3; may lower ALT or AST; may increase serum adiponectin; may lower plasma LDL; may increase plasma HDL; may lower plasma triglycerides; or any combination thereof.

[0080] The term "effective amount" may be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0081] "Flat dose" in the context of the disclosed methods and dosages refers to a dose administered to a patient without considering the patient's weight or body surface area (BSA). Thus, a flat dose is not administered as mg dose per kg, but as an absolute amount of molecule, e.g., mg. The term "weight-based dose" as used herein in contrast to a flat dose means that the dose administered to a patient is calculated based on the patient's weight. In some embodiments, the amount of FGF-21 polypeptide in the flat doses disclosed herein is based on Slope Spectroscopy, e.g., Agilent Cary 60 UV-Vis Spectrometer with SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0082] FGF-21 activity: The term "FGF-21 activity" refers to at least one biological activity of an FGF-21 polypeptide within an FGF-21 conjugate (e.g., a PEGylated FGF-21 conjugate of the present disclosure). The term "biological activity" refers to the ability of a molecule, e.g., an FGF-21 polypeptide (e.g., an FGF-21 conjugate or an FGF-21 polypeptide within an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4), to affect any physical or biochemical property of a biological system, biological pathway, biological molecule, or biological interaction of an organism, including, but not limited to, a virus, a bacteriophage, a transposon, a prion, an insect, a fungus, a plant, an animal, and a human.

[0083] For example, biological activity includes any of the biological functions performed by wild-type FGF-21. Exemplary methods for determining whether a molecule has at least one biological activity of wild-type FGF-21 (such as the wild-type FGF-21 polypeptide of SEQ ID NO: 3) can include any functional assay known in the art, including the methods disclosed in Examples 5 and 17 of U.S. Application Publication No. 2017 / 0189486, which is incorporated herein by reference in its entirety.

[0084] Reference compound: The relative level of biological activity is, for example, a polypeptide that has biological activity for the intended therapeutic use or has sufficiently high biological activity for this purpose, in comparison with a reference compound, e.g., the EC of the reference compound. 50 EC less than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than 50 The relative level of biological activity can be determined to identify polypeptides having an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, that have biological activity for the intended therapeutic use or that have sufficiently high biological activity for this purpose, e.g., the EC of the corresponding FGF-21 in a reference formulation. 50 EC less than 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than 50 The formulation may also be determined in relation to a reference formulation to identify formulations having

[0085] The reference compound described herein may be the corresponding FGF-21 sequence that lacks modification such as PEGylation described herein. For example, the reference compound may be a wild-type FGF-21 polypeptide sequence or a mutant FGF-21 polypeptide sequence (e.g., wild-type FGF-21 with amino acid substitution such as replacement of Q109 with a non-natural amino acid such as para-acetyl-L-phenylalanine), but without a PEG-conjugated moiety. Thus, in some embodiments, the reference compound may contain at least one non-natural amino acid that is not linked to a PEG moiety described herein. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0086] Exemplary reference compounds for the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, include, without limitation, wild-type FGF-21 of SEQ ID NO:3, and mutant FGF-21 polypeptide of SEQ ID NO:1.

[0087] In some embodiments, the reference compound may contain additional amino acid substitutions, deletions, and / or insertions. In some embodiments, the comparison may be performed with a PEGylated or non-PEGylated form of the polypeptide; in the former case, the comparison may be performed with a polypeptide that includes a non-natural amino acid or with a polypeptide that does not include a non-natural amino acid.

[0088] Identity: As used herein, the term "identity" refers to the overall conservation of monomers between polymer molecules, e.g., between polypeptide molecules. The term "identical" without further qualifiers, e.g., protein A is identical to protein B, implies that the sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical" is synonymous with describing them as having, e.g., "70% sequence identity."

[0089] The calculation of the percent identity of two polypeptide sequences can be carried out, for example, by aligning the two sequences for optimal comparison (e.g., for optimal alignment, gaps may be introduced into one or both of the first and second polypeptide sequences, and non-identical sequences may be discarded for comparison).In certain embodiments, the length of the sequence that is aligned for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.The amino acids at corresponding amino acid positions are then compared.

[0090] If a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, the molecules are identical at this position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap. The comparison of sequences between two sequences and the determination of percent identity can be achieved using a mathematical algorithm.

[0091] Suitable software programs are available from various sources and are software programs for both protein and nucleotide sequence alignment. One program suitable for determining sequence identity percentage is bl2seq, which is part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. Government's National Center for Biotechnology Information. bl2seq uses either the BLASTN or BLASTP algorithm to perform comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite, and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa. Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (Clustal W, Clustal X, or Clustal Omega), MUSCLE, etc.

[0092] Different regions in a single polypeptide target sequence that aligns with a polypeptide reference sequence can each have their own sequence identity percentage.It is noted that the sequence identity percentage value is rounded to the nearest tenth.For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2.It is also noted that the length value is always an integer.

[0093] In certain embodiments, the percent identity (ID%) of a first amino acid sequence to a second amino acid sequence is calculated as ID%=100×(Y / Z), where Y is the number of amino acid residues scored as identical matches in an alignment of the first sequence with the second sequence (aligned by visual inspection or by a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0094] Those skilled in the art will recognize that the generation of sequence alignment for calculating sequence identity percentage is not limited to the two-way sequence comparison driven solely by primary sequence data. It will also be recognized that sequence alignment can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., protein structure by crystallography), functional data (e.g., mutation location), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignment is T-Coffee, available at www.tcoffee.org, or alternatively available from, for example, EBI. It will also be recognized that the final alignment used to calculate sequence identity percentage can be automatically curated or manually curated.

[0095] In vivo proteolysis: The term "in vivo proteolysis" refers to the cleavage of a polypeptide when introduced into a biological system (e.g., when injected into an organism), which may result from proteases occurring within the organism. Proteolysis may potentially affect the biological activity or half-life of a polypeptide. For example, wild-type FGF-21 may undergo cleavage at the C-terminus, resulting in a truncated, inactive polypeptide.

[0096] An exemplary method for measuring in vivo proteolysis of FGF-21 is the Meso Scale Discovery (MSD)-based ECLIA (electrochemiluminescent immunosorbent assay), described in Example 10 of US Patent Publication No. 2017 / 0189486. The relative amount of in vivo or in vitro proteolysis can be determined, for example, against a reference compound to identify a polypeptide that reduces in vivo proteolysis. The relative amount of in vivo proteolysis can also be determined against a reference formulation, i.e., to identify a formulation in which the FGF-21 polypeptide portion of the FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, reduces in vitro proteolysis.

[0097] Isolated: As used herein, the term "isolated" refers to a material or entity (e.g., a polypeptide) that has been separated from at least some of the components with which it is associated (whether in nature or under experimental settings). Isolated materials (e.g., proteins) may have varying levels of purity relative to the materials with which they are associated.

[0098] Linked: The terms "linked," "fused," "conjugated," and "conjugated" are used interchangeably and refer to a covalently fused or concatenated PEG moiety (e.g., a PEG moiety of about 30 kD) that includes an insertion within an FGF-21 polypeptide, e.g., a mutant FGF-21 polypeptide disclosed herein (e.g., the FGF-21 polypeptide of SEQ ID NO:1).

[0099] In the context of the present disclosure, an FGF-2 polypeptide, such as a mutant FGF-2 polypeptide disclosed herein (e.g., the FGF-21 polypeptide of SEQ ID NO:1), and a PEG moiety may be "fused" as a result of chemical synthesis.

[0100] In the context of the present disclosure, the term "conjugate" or "conjugation" denotes that two molecular entities (e.g., an FGF-21 polypeptide, e.g., a mutant FGF-21 polypeptide disclosed herein, and a polymer moiety such as PEG) are chemically linked. In some specific embodiments, the FGF-21 polypeptide and the PEG moiety are linked via an oxime linkage, as shown in Formula I disclosed herein. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0101] Mutation: In the context of the present disclosure, the terms "mutation" and "amino acid substitution" (sometimes also simply referred to as "substitution"), as defined above, are considered to be interchangeable.

[0102] Unnatural Amino Acid: "Unnatural Amino Acid" refers to an amino acid that is not one of the 20 common amino acids, or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid", "unnatural amino acid", "non-naturally occurring amino acid", and various hyphenated and non-hyphenated forms thereof.

[0103] The term "unnatural amino acid" also includes, but is not limited to, amino acids that arise by modification (e.g., post-translational modification) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids), but that are not themselves naturally incorporated into a growing polypeptide chain by the translation complex. Examples of such unnatural amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In a specific embodiment of the disclosure, the unnatural amino acid is para-acetyl-phenylalanine. In a specific embodiment of the disclosure, the unnatural amino acid is para-acetyl-L-phenylalanine. In a specific embodiment of the disclosure, the unnatural amino acid is para-acetyl-D-phenylalanine.

[0104] Patient: As used herein, "patient" refers to a subject who may seek or need treatment, who requests treatment, who is receiving treatment, who is to receive treatment, or who is under the care of a professional trained in a particular disease or condition.

[0105] Pharmaceutical composition: The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of an active ingredient (e.g., an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4) to be effective, and does not contain additional ingredients (e.g., excipients and water) that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0106] Pharmaceutically acceptable: As used herein, the phrase "pharmacologically acceptable" is used to refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment and suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Approval by a federal or state regulatory agency (or the United States Pharmacopeia or other generally recognized pharmacopoeias) for use in animals in general, and more specifically for use in humans, implies that these compounds, materials, compositions, and / or dosage forms are pharmacopoeia-approved. Compounds, materials, compositions, and / or dosage forms that are generally acceptable as safe for therapeutic purposes are "therapeutically acceptable."

[0107] Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutical acceptable excipient" refers to any component other than the active compound described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that has substantially non-toxic and non-inflammatory properties in subjects. Excipients can include, for example, chelating agents, surfactants, buffers, osmotic pressure regulators, antioxidants, emulsifiers, bulking agents (diluents), preservatives, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. An excipient that is generally accepted as safe for therapeutic purposes is a "therapeutically acceptable excipient".

[0108] Pharmaceutically acceptable salts: The present disclosure also includes pharma- ceutically acceptable salts of the compounds described herein. As used herein, "pharma- ceutically acceptable salts" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base with a suitable organic acid). Examples of pharma- ceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.

[0109] Polypeptide: As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to polymers of amino acids of any length. The polymers may include modified amino acids. The terms also encompass amino acid polymers that are naturally or interveningly modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling moiety. Also included within the definition are polypeptides that contain, for example, one or more analogs of amino acids (including, for example, unnatural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art. In certain aspects, the polypeptides disclosed herein are FGF-21 polypeptides.

[0110] The term polypeptide as used herein refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single polypeptide or a multi-molecular complex, such as a dimer, trimer, or tetramer. A polypeptide may also include single-chain or multi-chain polypeptides. Most commonly, disulfide linkages are found in multi-chain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some aspects, a "peptide" may be 50 amino acids or less in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0111] Preventing: As used herein, the term "preventing" refers to partially or completely delaying the onset of a disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical symptoms of a particular disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or symptoms of a particular disease, disorder, and / or condition; partially or completely delaying the progression from a particular disease, disorder, and / or condition; and / or reducing the risk of developing pathologies associated with a disease, disorder, and / or condition. In some embodiments, the pharmaceutical formulations disclosed in the present application can be used to prevent the onset of, prevent symptoms of, or prevent complications of, diseases or conditions associated with fibrosis, such as NASH or diabetes.

[0112] Prophylactic: As used herein, "prophylactic" refers to a treatment or course of action used to prevent the onset of a disease or condition, or to prevent or delay the onset of a disease or condition associated with fibrosis, such as symptoms of NASH. In some aspects, the pharmaceutical preparations disclosed in the present application can be used prophylactically.

[0113] Prevention: As used herein, "prevention" refers to measures taken to maintain health, prevent or delay the onset of a disease or condition associated with fibrosis, such as NASH or diabetes, or prevent or delay the onset of symptoms associated with a disease or condition.

[0114] Recombinant: A "recombinant" polypeptide or protein refers to a polypeptide or protein produced through recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered to be isolated for the purposes of this disclosure, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. The mutant FGF-21 disclosed herein may be recombinantly produced using methods known in the art. The proteins and peptides disclosed herein may also be chemically synthesized.

[0115] In some aspects, FGF-21 polypeptides useful in the conjugates disclosed herein (e.g., FGF-21 polypeptides comprising unnatural amino acids, e.g., FGF-21 of SEQ ID NO:1) are recombinantly produced by a bacterial host.

[0116] Similarity: As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polypeptide molecules. Calculation of the percent similarity of polymer molecules to each other can be performed in the same manner as calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as understood in the art.

[0117] Soluble: The term "soluble" refers to the amount of a substance that can be dissolved in another substance, e.g., the amount of wild-type FGF-21 (e.g., an FGF-21 polypeptide of SEQ ID NO: 3), mutant FGF-21 (e.g., an FGF-21 polypeptide comprising a non-natural amino acid, e.g., an FGF-21 polypeptide of SEQ ID NO: 1), or an FGF-21 conjugate of the present disclosure (e.g., an FGF-21 polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4) that can be dissolved in an aqueous solution.

[0118] An exemplary method for measuring the solubility of wild-type FGF-21 polypeptide, mutant FGF-21 polypeptide, or FGF-21 conjugate is the plug flow solubility test described in Example 8 of US Patent Publication No. 2017 / 0189486. Relative solubility can be determined, for example, against a reference compound to identify a polypeptide that has increased solubility. In some embodiments, relative solubility can be determined against a reference formulation to identify a formulation in which the polypeptide has increased solubility.

[0119] Subject: "Subject" or "individual" or "animal" or "mammal" refers to any subject, particularly a mammalian subject, for which diagnosis, prognosis, or treatment is desired. Mammalian subjects include, but are not limited to, livestock, farm animals, zoo animals, sport animals, and pet animals, such as humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle; primates, such as apes, monkeys, orangutans, and chimpanzees; canines, such as dogs and wolves; felines, such as cats, lions, and tigers; equines, such as horses, donkeys, and zebras; bears; food animals, such as cows, pigs, and sheep; ungulates, such as deer and giraffes; rodents, such as mice, rats, hamsters, and guinea pigs, and the like. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In certain embodiments, the subject is a human patient or cells thereof suitable for the methods described herein, whether in vivo, in vitro or ex vivo.

[0120] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed as having or exhibiting one or more symptoms of the disease, disorder, and / or condition. In some embodiments, the pharmaceutical formulations disclosed herein may be administered to a subject suffering from a disease or condition associated with fibrosis, such as NASH or diabetes.

[0121] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition may not have been diagnosed with and / or may not exhibit symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the onset of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the onset of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein and / or nucleic acid associated with the onset of the disease, disorder, and / or condition; (4) a habit and / or lifestyle associated with the onset of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection with a microorganism associated with the onset of the disease, disorder, and / or condition.

[0122] In some embodiments, the individual who is susceptible to disease, disorder, and / or condition will develop the disease, disorder, and / or condition.In some embodiments, the individual who is susceptible to disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.In some embodiments, the pharmaceutical formulation disclosed herein can be administered to a subject who is susceptible to disease or condition associated with fibrosis, such as NASH or diabetes.

[0123] Therapeutic agent: The term "therapeutic agent" or "drug" refers to a molecular entity that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. For example, in some embodiments, an FGF-21 conjugate disclosed herein (e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4) can be a therapeutic agent. In some embodiments, the drug is another molecule that is co-administered as part of a combination therapy with at least one FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0124] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of delivered agent (e.g., an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, or a formulation comprising an FGF-21 conjugate) sufficient to treat, ameliorate symptoms, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the infection, disease, disorder, and / or condition. In general, administration of a therapeutically effective amount is expected to result in a therapeutically effective outcome.

[0125] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means an outcome sufficient to treat, ameliorate symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0126] Treat, Treatment, Therapy: As used herein, the terms "treat" or "treatment" or "therapy", or grammatical variations thereof, refer to partially or completely preventing, alleviating, ameliorating, improving, alleviating, delaying the onset of, inhibiting the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms or characteristics of a disease or condition associated with fibrosis, such as NASH or diabetes. For example, "treating" a disease associated with fibrosis may refer to preventing symptoms, ameliorating symptoms, delaying the onset of the disease or condition or its symptoms, etc. Treatment may be administered to subjects who do not exhibit symptoms of the disease, disorder, and / or condition and / or to subjects who exhibit only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0127] ug, uM, uL: As used herein, the terms "ug", "uM", and "uL" are used interchangeably with "μg", "μM", and "μL", respectively.

[0128] Various aspects of the disclosure are described in further detail in the following subsections. I. FGF-21 Conjugate Preparations

[0129] The present disclosure provides a pharmaceutical formulation comprising a fibroblast growth factor 21 (FGF-21) conjugate and an aminopolycarboxylic acid cation chelator, e.g., diethylenetriaminepentaacetic acid (DTPA), where the formulation has improved stability compared to a reference formulation. The term "reference formulation" refers to a formulation that contains the same ingredients and properties (e.g., pH, temperature) as a "test formulation" (a formulation that is compared to the reference formulation), except that it does not contain an aminopolycarboxylic acid cation chelator.

[0130] The term "FGF-21 conjugate" refers to a conjugate that includes an FGF-21 polypeptide moiety linked to a PEG moiety. In some embodiments, the PEG moiety includes a distal methoxy (-O-CH3) group. The term "FGF-21 polypeptide" generally refers to both wild-type FGF-21 polypeptides (e.g., the polypeptide of SEQ ID NO: 3) and "mutant FGF-21 polypeptides."

[0131] As used herein, the term "PEG-FGF-21 conjugate" or "PEG-FGF-21" refers to a PEGylated form of FGF-21 that includes a PEG moiety linked to a mutant FGF-21 polypeptide moiety via an oxime linkage. Exemplary PEG-FGF-21 conjugates of the present disclosure are set forth in SEQ ID NO: 2 or 5, and SEQ ID NO: 4 or 6 in Table 4 below. [Table 1]

[0132] Many FGF-21 known in the art can be used in the PEGylated FGF-21 conjugate formulation disclosed herein, for example, the PEGylated FGF-21 conjugate formulation disclosed in U.S. Patent No. 8,012,931 and U.S. Patent No. 9,434,788, each of which is incorporated herein by reference in its entirety.Fibroblast growth factor 21 (FGF-21) has been described in the literature (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206 (2000); WO01 / 36640 and WO01 / 18172; and U.S. Patent Publication No. 20040259780, each of which is incorporated herein by reference in its entirety). Unlike other FGFs, FGF-21 has been reported to have no proliferative or tumorigenic effects (Ornitz and Itoh, Genome Biology 2001, 2(3):reviews3005.1-3005.12).

[0133] In some embodiments, an FGF-21 polypeptide useful in the present disclosure comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 without the methionine at the N-terminus.

[0134] Several polymorphisms have been identified for FGF-21. In US Patent Publication No. 20010012628 and US Patent Publication No. 6,716,626, leucine or proline is described at the same position. N-terminal leader or signal sequences that differ by one amino acid (leucine) are shown in US Patent Publication No. 6,716,626 and US Patent Publication No. 20040259780. FGF-21 variants or mutants can be prepared using methods described in U.S. Pat. No. 6,716,626; U.S. Patent Publication Nos. 2005 / 0176631, 2005 / 0037457, 2004 / 0185494, 2004 / 0259780, 2002 / 0164713, and 2001 / 0012628; WO01 / 36640; WO03 / 011213; WO03 / 059270; WO04 / 110472; WO05 / 06 1712;WO05 / 072769;WO05 / 091944;WO05 / 113606;WO06 / 028595;WO06 / 028714;WO06 / 050247;WO06 / 065582;WO06 / 078463;WO01 / 018172;WO09 / 149171;WO10 / 042747;WO12 / 066075;WO11 / 154349;WO13 / 052311;WO13 / 188181.

[0135] As used herein, the terms "mutant FGF-21" and "mutant FGF-21 polypeptide" refer to FGF-21 polypeptides that differ from a reference wild-type FGF-21 polypeptide (e.g., wild-type human FGF-21 of SEQ ID NO: 3) at at least one amino acid position and typically have at least one biological activity of fibroblast growth factor 21, as well as FGF-21 analogs, FGF-21 isoforms, FGF-21 mimetics, FGF-21 fragments, hybrid FGF-21 proteins, fusion proteins, oligomers and multimers thereof, homologs, glycosylation pattern variants, splice variants, and muteins, regardless of their biological activity. The term "mutant FGF-21" encompasses both naturally occurring variants and non-naturally occurring variants, such as variants resulting from the substitution of an amino acid in a wild-type FGF-21 polypeptide, e.g., the polypeptide of SEQ ID NO: 3, with a non-natural amino acid (e.g., para-acetyl-L-phenylalanine). The substitution may be the result of, for example, recombinant expression, or chemical or enzymatic synthesis. In some embodiments, a mutant FGF-21 polypeptide of the present disclosure comprises, consists of, or consists essentially of the polypeptide of SEQ ID NO:1.

[0136] The mutant FGF-21 polypeptide of the present disclosure includes FGF-21 polypeptides that contain one or more amino acid substitutions, additions, or deletions. For example, the mutant FGF-21 polypeptide of the present disclosure includes modifications such as one or more amino acid substitutions (e.g., with naturally occurring or non-naturally occurring amino acids), deletions (terminal or internal deletions), or joining of heterologous moieties (joining at the C-terminus, N-terminus, or internally, either by insertion / insertion, or by side chain joining within the amino acid sequence). The term mutant FGF-21 polypeptide also includes polymorphisms of FGF-21 (e.g., sequence variants of naturally occurring FGF-21), such as P-form or L-form.

[0137] Substitutions at a wide variety of amino acid positions within naturally occurring FGF-21 polypeptides have been described. Substitutions that modulate solubility or stability, increase agonist activity, increase half-life in vivo or in vitro, increase protease resistance, convert the polypeptide into an antagonist, reduce immunogenicity or toxicity, facilitate purification, or promote manufacturability, or any combination thereof, are also encompassed by the term mutant FGF-21 polypeptide.

[0138] The term mutant FGF-21 polypeptide also includes biologically active fragments, biologically active variants, and stereoisomers of naturally occurring FGF-21 polypeptides, as well as agonist, mimetic, and antagonist variants of naturally occurring FGF-21, and polypeptide fusions thereof. Fusions containing additional amino acids at the amino terminus, carboxyl terminus, or both, are encompassed by the term mutant FGF-21 polypeptide.

[0139] Exemplary fusions include, but are not limited to, methionyl FGF-21 (wherein a methionine is linked to the N-terminus of FGF-21 polypeptide, e.g., obtained from recombinant expression of a mature form of FGF-21, or a portion thereof, lacking a leader or signal peptide), fusions for purification purposes (including, but not limited to, polyhistidine or affinity epitopes).

[0140] The term mutant FGF-21 polypeptide also includes glycosylated FGF-21 polypeptide, such as, but not limited to, polypeptide glycosylated at any amino acid position, polypeptide N-linked glycosylation form, or O-linked glycosylation form. Mutants containing single nucleotide changes are also considered biologically active mutants of FGF-21. In addition, splice variants are also included.

[0141] The term mutant FGF-21 polypeptide also includes heterodimers, homodimers, heteromultimers, or homomultimers of FGF-21 with any one or more unmodified or modified FGF-21, or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide analogs that contain, for example, specific deletions or other modifications, but still maintain biological activity.

[0142] In some embodiments, mutant FGF-21 polypeptides include additions, substitutions, or deletions that increase the affinity of the FGF-21 polypeptide for its receptor. Similarly, the term mutant FGF-21 polypeptide includes chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or polyHis) or other affinity-based sequences (including but not limited to FLAG, polyHis, GST, etc.) or linking molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification, transport across tissue or cell membranes, prodrug release or activation, reduction in FGF-21 polypeptide size, or other properties of the polypeptide.

[0143] In some embodiments, mutant FGF-21 polypeptides include polypeptides having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, or SEQ ID NO:1 or SEQ ID NO:3 without a methionine at the N-terminus, where the polypeptide has FGF-21 activity. In some embodiments, mutant FGF-21 polypeptides include the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0144] In some embodiments, the mutant FGF-21 polypeptide consists or consists essentially of a polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to SEQ ID NO:1 or SEQ ID NO:3, or the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 without a methionine at the N-terminus, where the polypeptide has FGF-21 activity. In some embodiments, the mutant FGF-21 polypeptide consists essentially of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6.

[0145] Mutant FGF-21 polypeptides encompassed by this definition include, for example, mutant FGF-21 polypeptides that contain at least one unnatural amino acid. In some embodiments, the unnatural amino acid is an amino acid that can form a stable oxime linkage when reacted with an aminooxy derivative, such as p-acetylphenylalanine, m-acetylphenylalanine, p-(3-oxobutenoyl)-L-phenylalanine, p-(2-amino-3-hydroxyethyl)phenylalanine, etc. In some embodiments, the unnatural amino acid is p-acetylphenylalanine. In some embodiments, the unnatural amino acid is p-acetyl-L-phenylalanine.

[0146] In some embodiments, the one or more unnatural amino acids are located at the following positions in wild-type FGF-21: before position 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 10 5, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 1 51, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., at the carboxyl terminus of the protein) (amino acid positions corresponding to SEQ ID NO:3).

[0147] In some specific aspects, the mutant FGF-21 of the present disclosure is a modified FGF-21 polypeptide of SEQ ID NO: 1, i.e., a derivative of wild-type FGF-21 of SEQ ID NO: 3, in which glutamine 109 of wild-type FGF-21 is replaced by the unnatural amino acid para-acetyl-L-phenylalanine.

[0148] In some specific aspects, the FGF-21 polypeptides of the present disclosure are modified such that the methionine at the N-terminus of the FGF-21 polypeptide (SEQ ID NO:1 or SEQ ID NO:3) is removed: SEQ ID NO:5 or 6.

[0149] As disclosed above, the mutant FGF-21 polypeptides of the present disclosure can be linked to a PEG (polyethylene glycol) moiety. Linking PEG to the mutant FGF-21 polypeptides disclosed herein (e.g., the FGF-21 polypeptide of SEQ ID NO: 1) can result in changes including, but not limited to, extending or modulating serum (in vivo) half-life or therapeutic half-life, modulating immunogenicity or toxicity, modulating physical association characteristics such as aggregation and multimerization, altering binding to receptors, altering binding to one or more binding partners, and altering receptor dimerization or multimerization, compared to the unmodified form. In some embodiments, the linkage of PEG to a mutant FGF-21 polypeptide disclosed herein (e.g., FGF-21 polypeptide of SEQ ID NO: 1) improves or alters the pharmacokinetic or biophysical properties, including but not limited to, increased absorption rate, reduced toxicity, improved solubility, reduced protein aggregation, increased biological activity and / or target selectivity of PEGylated FGF-21, increased manufacturability, and / or reduced immunogenicity (see, e.g., U.S. Pat. No. 4,179,337), compared to a reference compound, such as a mutant FGF-21 polypeptide (e.g., FGF-21 polypeptide of SEQ ID NO: 1) or an unconjugated form of wild-type FGF-21 (e.g., FGF-21 polypeptide of SEQ ID NO: 3). In some embodiments, at least one linker may be interposed between the mutant FGF-21 polypeptide moiety and the PEG moiety. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0150] Examination of the crystal structures of FGF-21 or FGF family members and their interactions with the FGF receptors can indicate which particular amino acid residues have side chains that are fully or partially accessible to the solvent. The side chains of unnatural amino acids at these positions point away from the protein surface and towards the external solvent and can therefore be linked to PEG.

[0151] PEG may be present at the following amino acid positions of a wild-type or mutant FGF-21 polypeptide: before position 1 (i.e., at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 10 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, PEG can be linked to one or more of: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 (i.e., the carboxyl terminus of the protein) (amino acid positions corresponding to SEQ ID NO:3). In some embodiments, PEG is conjugated to the side chain of a non-natural amino acid, e.g., a phenylalanine derivative, such as para-acetyl-L-phenylalanine, replacing a naturally occurring amino acid at any of the positions disclosed above.

[0152] The PEG of the present disclosure is polyethylene glycol, polyethylene glycol propionaldehyde, or any of C1-C 10 Monoalkoxy derivatives or C1-C 10These include, but are not limited to, monoaryloxy derivatives (described in U.S. Pat. No. 5,252,714, which is incorporated herein by reference), monomethoxypolyethylene glycol, discrete PEG, polypropylene oxide / ethylene oxide copolymer, polyalkylene glycol and its derivatives, copolymers of polyalkylene glycol and its derivatives, or mixtures thereof. In some embodiments, PEG can have a branched structure. Branched PEGs are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999).

[0153] In some embodiments, the molecular weight of PEG is about 30 kDa. Other sizes may be used depending on the desired profile (e.g., the desired duration of sustained release, the effect on biological activity, if any, ease of manipulation, the degree or lack of antigenicity, and other known effects of polyethylene glycol on proteins or analogs). In some embodiments, the molecular weight of PEG is about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, or about 32 kDa. In some embodiments, the molecular weight of PEG is between about 28 kDa and about 29 kDa, between about 29 kDa and about 30 kDa, between about 30 kDa and about 31 kDa, or between about 31 kDa and about 32 kDa. Methods for determining the molecular weight of PEG (as well as FGF-21 conjugates) are well known in the art. Any known method can be used to measure the molecular weight of PEG for the purposes of this disclosure, such as mass spectrometry (e.g., MALDI-TOF and / or ESI).

[0154] In some embodiments, the PEG has about 600 ethylene glycol units, about 610 ethylene glycol units, about 620 ethylene glycol units, about 630 ethylene glycol units, about 640 ethylene glycol units, about 650 ethylene glycol units, about 660 ethylene glycol units, about 670 ethylene glycol units, about 680 ethylene glycol units, about 690 ethylene glycol units, about 700 ethylene glycol units, about 710 ethylene glycol units, about 720 ethylene glycol units, about 730 ethylene glycol units, about 740 ethylene glycol units, about 750 ethylene glycol units, about 760 ethylene glycol units, about 770 ethylene glycol units, about 780 ethylene glycol units, about 790 ethylene glycol units, or about 800 ethylene glycol units.

[0155] In some embodiments, the PEG is between about 600 ethylene glycol units and about 610 ethylene glycol units, between about 610 ethylene glycol units and about 620 ethylene glycol units, between about 620 ethylene glycol units and about 630 ethylene glycol units, between about 630 ethylene glycol units and about 640 ethylene glycol units, between about 640 ethylene glycol units and about 650 ethylene glycol units, between about 650 ethylene glycol units and about 660 ethylene glycol units, between about 660 ethylene glycol units and about 670 ethylene glycol units, between about 670 ethylene glycol units and about 680 ethylene glycol units, between about 680 ethylene glycol units and about 690 ethylene glycol units, between about 690 ethylene glycol units and about 700 ethylene glycol units between about 700 ethylene glycol units and about 710 ethylene glycol units, between about 710 ethylene glycol units and about 720 ethylene glycol units, between about 720 ethylene glycol units and about 730 ethylene glycol units, between about 730 ethylene glycol units and about 740 ethylene glycol units, between about 740 ethylene glycol units and about 750 ethylene glycol units, between about 750 ethylene glycol units and about 760 ethylene glycol units, between about 760 ethylene glycol units and about 770 ethylene glycol units, between about 770 ethylene glycol units and about 780 ethylene glycol units, between about 780 ethylene glycol units and about 790 ethylene glycol units, or between about 790 ethylene glycol units and about 800 ethylene glycol units.

[0156] In some embodiments, the PEG has 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, or 700 ethylene glycol units.

[0157] In some specific embodiments, the PEG moiety is linked to a mutant FGF-21 polypeptide of the present disclosure (e.g., the FGF-21 polypeptide of SEQ ID NO: 1) via an oxime linkage. In some embodiments, the PEG moiety is linked to a reactive group of the PEG molecule (e.g., the 30 kDa methyl PEG provided in FIG. 7). 681 The PEG moiety is linked to a mutant FGF-21 polypeptide of the disclosure (e.g., a mutant FGF-21 polypeptide of SEQ ID NO: 1) via an oxime linkage formed between the aminooxy group of the aminooxy molecule and a reactive group of an unnatural amino acid in the mutant FGF-21 polypeptide (e.g., the acetyl group of p-acetyl-phenylalanine at amino acid position 109 of the mutant FGF-21 polypeptide sequence). In some embodiments, the unnatural amino acid is para-acetyl-L-phenylalanine, replacing Gln109 of SEQ ID NO: 3. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0158] In some embodiments, the FGF-21 conjugate in the formulations disclosed herein is PEG-FGF-21 of SEQ ID NO: 2, i.e., FGF-21 of SEQ ID NO: 1, in which glutamine 109 (SEQ ID NO: 3) of wild-type FGF-21 is replaced with para-acetyl-L-phenylalanine, and a PEG moiety, e.g., a linear PEG of between about 28 kDa and about 32 kDa molecular weight, e.g., about 30 kDa, is covalently attached to the para-acetyl-L-phenylalanine via an oxime linkage. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0159] In some embodiments, the FGF-21 conjugate in the formulation disclosed herein is a PEG-FGF-21 of SEQ ID NO:2, i.e., an FGF-21 conjugate comprising (i) an FGF-21 of SEQ ID NO:1, in which para-acetyl-L-phenylalanine replaces glutamine 109 (SEQ ID NO:3) of wild-type FGF-21, and (ii) a PEG moiety, e.g., a linear PEG of molecular weight between about 28 kDa and about 32 kDa, e.g., about 30 kDa, covalently attached to the para-acetyl-L-phenylalanine via an oxime linkage. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0160] In some embodiments, the FGF-21 conjugate in the formulation disclosed herein is PEG-FGF-21 of SEQ ID NO: 4, i.e., FGF-21 of SEQ ID NO: 1, in which glutamine 109 (SEQ ID NO: 3) of wild-type FGF-21 is replaced with para-acetyl-L-phenylalanine, and a PEG moiety comprising 681 ethylene glycol units is covalently attached to the para-acetyl-L-phenylalanine via an oxime linkage. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0161] In some embodiments, the FGF-21 conjugate in the formulation disclosed herein is PEG-FGF-21 of SEQ ID NO: 4, i.e., an FGF-21 conjugate comprising (i) an FGF-21 of SEQ ID NO: 1, in which para-acetyl-L-phenylalanine replaces glutamine 109 (SEQ ID NO: 3) of wild-type FGF-21, and (ii) a PEG moiety comprising 681 ethylene glycol units covalently bonded to the para-acetyl-L-phenylalanine via an oxime linkage. In some embodiments, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0162] Thus, in some specific aspects of the disclosure, the FGF-21 conjugate in the formulation disclosed herein comprises (i) an FGF-21 polypeptide moiety of SEQ ID NO: 1, and (ii) a PEG moiety with 681 ethylene glycol units, i.e., a molecular weight of about 30 kDa, covalently attached via an oxime linkage to para-acetyl-L-phenylalanine at position 109. In some aspects, the PEG moiety comprises a distal methoxy (-O-CH3) group.

[0163] In some embodiments, the oxime linkage is between a reactive group of an unnatural amino acid present in a mutant FGF-21 (e.g., the acetyl group of p-acetylphenylalanine) and a reactive group of a PEG molecule (e.g., the 30 kDa PEG containing the distal methoxy group depicted in FIG. 7). 681 The aminooxy group of an aminooxy molecule is formed by chemical reaction with the aminooxy group of the aminooxy molecule. In some embodiments, the unnatural amino acid can be incorporated into a mutant FGF-21 polypeptide recombinantly (e.g., by expression in a prokaryotic cell culture), using in vitro transcription and translation, using chemical synthesis, or using any method known in the art. In some embodiments, a PEG molecule is chemically linked to an amino acid (e.g., p-acetyl-phenylalanine), and the PEG-amino acid can then be incorporated into the FGF-21 polypeptide, for example, via chemical synthesis. In some embodiments, the PEG molecule comprises a distal methoxy (-O-CH3) group.

[0164] The production of FGF-21 polypeptides via yeast expression has previously been hindered by the presence of O-linked glycosylation, which requires site-directed mutagenesis to remove O-linked glycosylation sites. FGF-21 polypeptides also exhibit a substantial degree of glycosylation when recombinantly produced in mammalian cell culture. Thus, in some embodiments, the mutant FGF-21 polypeptides of the present disclosure are recombinantly produced in prokaryotic cell culture. Thus, in some embodiments, the mutant FGF-21 polypeptides of the present disclosure are not glycosylated.

[0165] As disclosed above, the present disclosure provides a pharmaceutical formulation comprising the FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, and an aminopolycarboxylic acid cation chelator, for example, diethylenetriaminepentaacetic acid (DTPA). The terms "chelator" or "cation chelator" are interchangeable and refer to any substance capable of removing metal ions from a solution system by forming new complex ions that have chemical properties different from those of the original metal ion. In particular, the cation chelator disclosed herein is a chelator that specifically binds to divalent metals, for example, Ca++.

[0166] In some embodiments, the FGF-21 conjugate formulations disclosed herein exhibit one or more stability improvements, such as a reduced deamidation rate and / or a reduced aggregation rate. For example, the incorporation of an aminopolycarboxylic acid cation chelator, such as DTPA, into an FGF-21 conjugate formulation can reduce the deamidation rate of the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, when stored at a certain temperature (e.g., 40°C) and for a certain period of time (e.g., one month), relative to a reference formulation.

[0167] In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or 5 or SEQ ID NO:4 or 6, in a pharmaceutical formulation stored at about 25° C., about 30° C., about 35° C., about 40° C., or about 45° C., relative to a reference formulation. In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or 5 or SEQ ID NO:4 or 6, in a pharmaceutical formulation stored at a temperature above 25° C., above 30° C., above 35° C., about 40° C., or about 45° C., relative to a reference formulation.

[0168] In some embodiments, an aminopolycarboxylate cation chelator (e.g., DTPA) may reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation stored at between about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, or about 40°C to about 45°C, relative to a reference formulation.

[0169] In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) may reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation after storage at a temperature or temperature range disclosed above for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or about 4 months relative to a reference formulation.

[0170] In a specific embodiment, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation stored at 40° C. for about one month relative to a reference formulation.

[0171] In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the high molecular weight (HMW) aggregation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation stored at about 25° C., about 30° C., about 35° C., about 40° C., or about 45° C., relative to a reference formulation. In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation stored at a temperature greater than 25° C., greater than 30° C., greater than 35° C., about 40° C., or about 45° C., relative to a reference formulation.

[0172] In some embodiments, an aminopolycarboxylate cation chelator (e.g., DTPA) may reduce the HMW aggregation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, in a pharmaceutical formulation stored at between about 20° C. to about 25° C., about 25° C. to about 30° C., about 30° C. to about 35° C., or about 40° C. to about 45° C., relative to a reference formulation.

[0173] In some embodiments, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, in a pharmaceutical formulation after storage for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or about 4 months at a temperature or temperature range disclosed above, relative to a reference formulation. In a specific embodiment, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, in a pharmaceutical formulation stored at 40° C. for about 1 month, relative to a reference formulation.

[0174] In some embodiments, an aminopolycarboxylic acid cation chelator, e.g., DTPA, prevents or mitigates oxidation of one or more amino acids, e.g., methionine, in an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, an aminopolycarboxylic acid cation chelator, e.g., DTPA, prevents or mitigates oxidation of amino acid 1 and / or amino acid 169 of SEQ ID NO: 3 (or the corresponding amino acid in SEQ ID NO: 1, 2, or 4) at 25°C and / or 40°C.

[0175] In some specific embodiments, the aminopolycarboxylic acid cation chelator is DTPA. Pentetic acid or diethylenetriaminepentaacetic acid (DTPA) is an aminopolycarboxylic acid consisting of a diethylenetriamine backbone with five carboxymethyl groups. The conjugate base of DTPA has a high affinity for metal cations. Thus, the pentanion DTPA 5- is each nitrogen center and each COO - Assuming that the group is viewed as a center for coordination bonding, it is potentially an octadentate ligand. The formation constant for the complex is about 100 times greater than that for EDTA.

[0176] As a chelating agent, DTPA envelops metal ions by forming up to eight bonds. However, transition metals typically form fewer than eight coordinate bonds. Thus, DTPA still has the ability to bind other reagents after complexing with metals, as demonstrated by its derivative, the pendetide. For example, in its complex with copper(II), DTPA binds in a hexadentate-like fashion, utilizing three amine centers and three of the five carboxylates.

[0177] In some other embodiments, the aminopolycarboxylic acid cation chelator can be another aminopolycarboxylic acid cation chelator, such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or related compounds, such as tiuxetan (a modified form of DTPA whose carbon skeleton contains isothiocyanatobenzyl and methyl groups). Other chelators related to DTPA and EDTA known in the art are those in which the nitrogen of the amide group is one or more C1-C 18 Alkyl group substituted chelating agents, for example, DTPA.BMA and EDTA.BMA.

[0178] In some embodiments, the DTPA cation chelator is present in an amount between about 10 μM and about 100 μM, between 15 μM and about 95 μM, between about 20 μM and about 90 μM, between about 25 μM and about 85 μM, between about 30 μM and about 80 μM, between about 35 μM and about 75 μM, between about 40 μM and about 70 μM, between about 45 μM and about 65 μM, between about 50 μM and about 60 μM, between about 25 μM and about 75 μM, between about 40 μM and about 60 μM, between about 30 μM and about 70 μM, or between about 40 μM and about 75 μM.

[0179] In some embodiments, the DTPA cation chelator is present in an amount of about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, or about 100 μM.

[0180] In some embodiments, the DTPA cation chelator is present in an amount of at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, at least about 55 μM, at least about 60 μM, at least about 65 μM, at least about 70 μM, or at least about 75 μM.

[0181] In a specific embodiment, the aminopolycarboxylic acid cation chelator, eg, DTPA, is present in an amount of 50 μM.

[0182] In some embodiments, the pH of the formulations disclosed herein is greater than about 6.5, greater than about 6.6, greater than about 6.7, greater than about 6.8, greater than about 6.9, greater than about 7.0, greater than about 7.1, greater than about 7.2, greater than about 7.3, greater than about 7.4, or greater than about 7.5. In some embodiments, the pH of the formulation is greater than 6.5, greater than 6.6, greater than 6.7, greater than 6.8, greater than 6.9, greater than 7.0, greater than 7.1, greater than 7.2, greater than 7.3, greater than 7.4, or greater than 7.5. In some embodiments, the pH of the formulation is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5.

[0183] In some embodiments, the pH of the formulation is between about 6.5 to about 7.5, about 6.6 to about 7.5, about 6.7 to about 7.5, about 6.8 to about 7.5, about 6.9 to about 7.5, about 7.0 to about 7.5, about 7.1 to about 7.5, about 7.2 to about 7.5, about 7.3 to about 7.5, about 7.4 to about 7.5, about 6.5 to about 7.4, about 6.5 to about 7.3, about 6.5 to about 7.2, about 6.5 to about 7.1, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.4, about 6.7 to about 7.4, about 6.8 to about 7.4, about 6.9 about 7.4, about 7.0 to about 7.4, about 7.1 to about 7.4, about 7.2 to about 7.4, about 7.3 to about 7.4, about 6.5 to about 7.3, about 6.6 to about 7.3, about 6.7 to about 7.3, about 6.7 to about 7.3, about 6.8 to about 7.3, about 6.9 to about 7.3, about 7.0 to about 7.3, about 7.1 to about 7.3, about 7.2 to about 7.3, about 6.5 to about 7.2, about 6.6 to about 7.2, about 6.7 to about 7.2, about 6.8 to about 7.2, about 6.9 to about 7.2, about 7.0 to about 7.2, about 7.1 to about 7.2, about 6.9 to about 7.1, or about 7.0 to about 7.1.

[0184] In some embodiments, the pH of the formulation is about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the pharmaceutical formulation is more stable than a reference formulation having a pH of 6.5.

[0185] In some embodiments, the pharmaceutical preparation further comprises surfactant.As used herein, the term "surfactant" refers to any compound, typically an aliphatic molecule, that when dissolved or suspended in water or an aqueous solution, reduces surface tension, or reduces the surface tension between two liquids, or between a liquid and a solid.

[0186] In the context of this disclosure, a surfactant is any compound that reduces the interfacial stress and shear in a solution containing an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6.

[0187] In some embodiments, the surfactant is a non-ionic surfactant, i.e., a surfactant that tends to have no net charge in neutral solution. In some embodiments, the non-anionic surfactant is a polysorbate. Polysorbates are an important class of non-ionic surfactants that are widely used in protein pharmaceuticals to stabilize proteins against interface-induced aggregation and minimize protein surface adsorption (Wang W 2005. Protein aggregation and its inhibition in biopharmaceutics. Int J Pharm 289 (1-2):1-30). Polysorbates are amphiphilic, non-ionic surfactants composed of fatty acid esters of polyoxyethylene (POE) sorbitan. Commercially available polysorbates are chemically diverse mixtures that mainly contain sorbitan POE fatty acid esters.

[0188] As used herein, the term "polysorbate" refers to oleic acid esters of sorbitol and its anhydrides, typically copolymerized with ethylene oxide. Exemplary polysorbates include polysorbate 20 (TWEEN 20; PS20) (polyoxyethylene (20) sorbitan monolaurate); polysorbate 40 (TWEEN 40; PS40) (polyoxyethylene (20) sorbitan monopalmitate); polysorbate 60 (TWEEN 60; PS60) (polyoxyethylene (20) sorbitan monostearate); and polysorbate 80 (TWEEN 80; PS80) (polyoxyethylene (20) sorbitan monooleate).

[0189] The number 20 following the "polyoxyethylene" portion refers to the number of oxyethylene -(CHCH 2O The "polysorbate" portion refers to the total number of fatty acid groups associated with the polyoxyethylene sorbitan portion of the molecule. Monolaurate is designated by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80. In some embodiments, the nonionic surfactant is present in an amount above the critical micelle concentration (CMC), which for polyoxyethylene sorbitan fatty acid esters is an amount of at least about 0.01 mg / ml. See Wan and Lee, Journal of Pharm Sci, 63, p.136, 1974. Throughout this specification, surfactant concentration (%) corresponds to (w / v).

[0190] In some embodiments, the polysorbate is polysorbate 80 (PS80). In some embodiments, the surfactant that is polysorbate 80 is about 0.01% to about 0.1% (w / v), about 0.02% to about 0.1% (w / v), about 0.03% to about 0.1% (w / v), about 0.04% to about 0.1% (w / v), about 0.05% to about 0.1% (w / v), about 0.06% to about 0.1% (w / v), about 0.07% to about 0.1% (w / v), about 0.08% to about 0.1% (w / v), about 0.09% to about 0.1% (w / v), about 0.02% to about 0.09% (w / v), about 0.03% to about 0.09% (w / v), about 0.04% to about 0.09 ... % ~ approx. 0.09% (w / v), approx. 0.06% ~ approx. 0.09% (w / v), approx. 0.07% ~ approx. 0.09% (w / v), approx. 0.08% ~ approx. 0.0 9% (w / v), about 0.03% to about 0.08% (w / v), about 0.04% to about 0.08% (w / v), about 0.05% to about 0.08% (w / v) ), about 0.06% to about 0.08% (w / v), about 0.07% to about 0.08% (w / v), about 0.04% to about 0.07% (w / v), about 0.05% to about 0.07% (w / v), about 0.06% to about 0.07% (w / v), or about 0.05% to about 0.06% (w / v).

[0191] In some embodiments, the surfactant that is polysorbate 80 is present in an amount of at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), or at least about 0.1% (w / v).

[0192] In some embodiments, the surfactant, e.g., polysorbate 80, mitigates the formation of particulates and / or bubbles, e.g., when the formulation is agitated on a shaker. In some embodiments, the presence of a surfactant, e.g., polysorbate 80, in the formulation may reduce particulate formation by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the particulate formation level in a reference formulation. In some embodiments, the presence of a surfactant, e.g., polysorbate 80, in the formulation may reduce air bubble formation by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the air bubble formation level in a reference formulation.

[0193] In some embodiments, the pharmaceutical formulation further comprises an amino acid buffer.Amino acids are naturally occurring substances that can be easily metabolized, so they can be advantageously used as buffers in pharmaceutical applications.In addition, the amino acids used as buffers can also protect proteins in amorphous phase when the formulation is lyophilized.Suitable amino acid buffers can contain histidine, lysine, and / or arginine.Histidine has good buffering capacity at about pH 7.

[0194] As used herein, the term "histidine" includes L-histidine or D-histidine, solvated forms of histidine, hydrated forms of histidine (e.g., monohydrate), or anhydrous forms of histidine, or mixtures thereof. Other suitable buffers in the formulations of the present disclosure are glutamic acid, tris, or succinic acid, to name a few.

[0195] In some specific aspects, the amino acid buffer is L-histidine.

[0196] In some embodiments, the histidine buffer is about 10 mM to about 100 mM histidine, about 15 mM to about 100 mM histidine, about 20 mM to about 100 mM histidine, about 25 mM to about 100 mM, about 30 mM to about 100 mM histidine, about 35 mM to about 100 mM histidine, about 40 mM to about 100 mM histidine, about 45 mM to about 100 mM histidine, about 50 mM to about 100 mM histidine. In some embodiments, the histidine may be present in an amount of about 55 mM to about 100 mM histidine, about 60 mM to about 100 mM histidine, about 65 mM to about 100 mM histidine, about 70 mM to about 100 mM histidine, about 75 mM to about 100 mM histidine, about 80 mM to about 100 mM histidine, about 85 mM to about 100 mM histidine, about 90 mM to about 100 mM histidine, or about 95 mM to about 100 mM histidine.

[0197] In some embodiments, the histidine buffer is from about 20 mM to about 90 mM histidine, from about 25 mM to about 90 mM histidine, from about 30 mM to about 90 mM histidine, from about 35 mM to about 90 mM histidine, from about 40 mM to about 90 mM histidine, from about 45 mM to about 90 mM histidine, from about 50 mM to about 90 mM histidine, from about 55 mM to about 90 mM histidine, from about 60 mM to about 90 mM histidine, about 65 mM to about 90 mM histidine, about 70 mM to about 90 mM histidine, about 75 mM to about 90 mM histidine, about 80 mM to about 90 mM histidine, about 85 mM to about 90 mM histidine, about 30 mM to about 80 mM histidine, about 35 mM to about 80 mM histidine, about 40 mM to about 80 mM histidine, about 45 mM to about 80 mM histidine.

[0198] In some embodiments, the histidine buffer is about 50 mM to about 80 mM histidine, about 55 mM to about 80 mM histidine, about 60 mM to about 80 mM histidine, about 65 mM to about 80 mM histidine, about 70 mM to about 80 mM histidine, about 75 mM to about 80 mM histidine, about 40 mM to about 70 mM histidine, about 45 mM to about 70 mM histidine, about 50 mM to about 70 mM histidine. In some embodiments, the histidine may be present in an amount of about 55 mM to about 70 mM histidine, about 60 mM to about 70 mM histidine, about 65 mM to about 70 mM histidine, about 10 mM to about 30 mM histidine, about 15 mM to about 30 mM histidine, about 20 mM to about 30 mM histidine, about 25 mM to about 30 mM histidine, about 15 mM to about 25 mM histidine, or about 20 mM to about 25 mM histidine.

[0199] In some embodiments, the histidine buffer is present in an amount of about 15 mM to about 20 mM histidine, about 40 mM to about 60 mM histidine, about 45 mM to about 60 mM histidine, about 50 mM to about 60 mM histidine, about 15.5 mM to about 24.5 mM histidine, about 16 mM to about 24 mM histidine, about 16.5 mM to about 23.5 mM histidine, about 17 mM to about 23 mM histidine, about 17.5 mM to about 22.5 mM histidine, about 18 mM to about 22 mM histidine, about 18.5 mM to about 21.5 mM histidine, about 19 mM to about 21 mM histidine, or about 19.5 mM to about 20.5 mM histidine.

[0200] In some embodiments, the histidine buffer is about 10 mM histidine, about 11 mM histidine, about 12 mM histidine, about 13 mM histidine, about 14 mM histidine, about 15 mM histidine, about 16 mM histidine, about 17 mM histidine, about 18 mM histidine, about 19 mM histidine, about 20 mM histidine, about 21 mM histidine, about 22 mM histidine, about 23 mM histidine, about 24 mM histidine, about 25 mM histidine, about 26 mM histidine, about 27 mM histidine, about 28 mM histidine, about 29 mM histidine, about Histidine is present in an amount of about 30 mM histidine, about 31 mM histidine, about 32 mM histidine, about 33 mM histidine, about 34 mM histidine, about 35 mM histidine, about 36 mM histidine, about 37 mM histidine, about 38 mM histidine, about 39 mM histidine, about 40 mM histidine, about 41 mM histidine, about 42 mM histidine, about 43 mM histidine, about 44 mM histidine, about 45 mM histidine, about 46 mM histidine, about 47 mM histidine, about 48 mM histidine, about 49 mM histidine, or about 50 mM histidine.

[0201] In some embodiments, the pharmaceutical formulation further comprises an osmotic agent (also known in the art as an isotonicity agent). In accordance with the present disclosure, the osmotic agent (isotonicity agent) may comprise a polyol, a sugar, a carbohydrate, a salt such as sodium chloride, or a mixture thereof. Exemplary polyols include polyols with a molecular weight of less than about 600 kD (e.g., in the range of 120-400 kD), such as mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or a mixture thereof.

[0202] Sugar or carbohydrate osmotic modifiers include monosaccharides, disaccharides, and polysaccharides, or mixtures thereof. In some embodiments, the sugar or carbohydrate is selected from the group consisting of fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water soluble glucan, and mixtures thereof.

[0203] In some aspects, the osmolality adjusting agent comprises a sugar selected from the group of reducing or non-reducing sugars, or mixtures thereof. In some aspects, the osmolality adjusting agent or tonicity agent comprises a sugar, which is preferably a non-reducing sugar, sugar, selected from the group consisting of sucrose, trehalose, or mixtures thereof. In some specific aspects, the non-reducing sugar is sucrose.

[0204] In some embodiments, the osmotic regulator that is sucrose is about 100 mM to about 1 M sucrose, about 200 mM to about 1 M sucrose, about 300 mM to about 1 M sucrose, about 400 mM to about 1 M sucrose, about 500 mM to about 1 M sucrose, about 600 mM to about 1 M sucrose, about 700 mM to about 1 M sucrose, about 800 mM to about 1 M sucrose, about 900 mM to about 1 M sucrose, about 200 mM to about 900 mM sucrose, about 300 mM to about 900 mM sucrose, about 400 mM to about 900 mM sucrose, about 500 mM to about 900 mM sucrose. The sucrose may be present in an amount of about 600 mM to about 900 mM sucrose, about 700 mM to about 900 mM sucrose, about 800 mM to about 900 mM sucrose, about 300 mM to about 800 mM sucrose, about 400 mM to about 800 mM sucrose, about 500 mM to about 800 mM sucrose, about 600 mM to about 800 mM sucrose, about 700 mM to about 800 mM sucrose, about 400 mM to about 700 mM sucrose, about 500 mM to about 700 mM sucrose, about 600 mM to about 700 mM sucrose, or about 500 mM to about 600 mM sucrose.

[0205] In some embodiments, the osmotic modifier that is sucrose is present in an amount of about 100 mM sucrose, about 150 mM sucrose, about 200 mM sucrose, about 250 mM sucrose, about 300 mM sucrose, about 350 mM sucrose, about 400 mM sucrose, about 450 mM sucrose, about 500 mM sucrose, about 550 mM sucrose, about 600 mM sucrose, about 650 mM sucrose, about 700 mM sucrose, about 750 mM sucrose, about 800 mM sucrose, about 850 mM sucrose, about 900 mM sucrose, about 950 mM sucrose, or about 1 M sucrose.

[0206] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, are present at a concentration between about 1 mg / ml and about 40 mg / ml. In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, are present at a concentration of about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, or about 40 mg / ml. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulations disclosed herein is based on Slope Spectroscopy, e.g., Agilent Cary 60 UV-Vis spectrometer equipped with SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0207] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., the PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, are present at a concentration of between about 1 mg / ml and 5 mg / ml, between about 5 mg / ml and about 10 mg / ml, between about 10 mg / ml and about 15 mg / ml, between about 15 mg / ml and about 20 mg / ml, between about 20 mg / ml and about 25 mg / ml, between about 25 mg / ml and about 30 mg / ml, between about 30 mg / ml and about 35 mg / ml, or between about 35 mg / ml and about 40 mg / ml.

[0208] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, have a concentration of about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 20 mg / ml, about 22 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml, about 31 mg / ml, about 32 mg / ml, about 33 mg / ml, about 34 mg / ml, about 35 mg / ml, about 36 mg / ml, about 37 mg / ml, about 38 mg / ml, about 39 mg / ml, about 40 mg / ml, about 41 mg / ml, about 42 mg / ml, about 43 mg / ml, about 44 mg / ml, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml, about 59 mg / ml, about 60 mg / ml, about 61 mg / ml, about 62 mg / ml In a specific embodiment, the FGF-21 conjugate disclosed herein, e.g., the PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, is present at a concentration of about 10 mg / ml. In a specific embodiment, the FGF-21 conjugate disclosed herein, for example, the PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is present at a concentration of about 20 mg / ml.

[0209] In some embodiments, the concentration of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is determined according to methods known in the art or specific methods disclosed in the Examples section herein.

[0210] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein in an amount between about 1 mg and about 40 mg per dose. In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein in an amount between about 1 mg and about 5 mg per dose, between about 1 mg and about 10 mg per dose, or between about 1 mg and about 15 mg per dose, between about 1 mg and about 20 mg per dose, or between about 1 mg and about 25 mg per dose, between about 1 mg and about 30 mg per dose, or between about 1 mg and about 35 mg per dose, or between about 1 mg and about 40 mg per dose. In some embodiments, the dose is a flat dose. In some embodiments, the amount of FGF-21 conjugate in the pharmaceutical formulations disclosed herein is based on Slope Spectroscopy, e.g., Agilent Cary 60 UV-Vis spectrometer equipped with SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0211] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein in an amount between about 1 mg and about 21 mg per dose, between about 1 mg and about 22 mg per dose, between about 1 mg and about 23 mg per dose, between about 1 mg and about 24 mg per dose, between about 1 mg and about 25 mg per dose, between about 1 mg and about 26 mg per dose, between about 1 mg and about 27 mg per dose, between about 1 mg and about 28 mg per dose, between about 1 mg and about 29 mg per dose, or between about 1 mg and about 30 mg per dose. In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein in high amounts of more than 40 mg per dose. In some embodiments, the dose is a flat dose.

[0212] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein at about 1 mg per dose, about 2 mg per dose, about 3 mg per dose, about 4 mg per dose, about 5 mg per dose, about 6 mg per dose, about 7 mg per dose, about 8 mg per dose, about 9 mg per dose, about 10 mg per dose, about 11 mg per dose, about 12 mg per dose, about 13 mg per dose, about 14 mg per dose, about 15 mg per dose, about 16 mg per dose, about 17 mg per dose, about 18 mg per dose, about 19 mg per dose, about 20 mg per dose, about 21 mg per dose, about 22 mg per dose, about 23 mg per dose, about 24 mg per dose, about 25 mg per dose, about 26 mg per dose, about 27 mg per dose, about 28 mg per dose, about 29 mg per dose, about 30 mg per dose, about 31 mg per dose, about 32 mg per dose, about 33 mg per dose, about 34 mg per dose, about 35 mg per dose, about 36 mg per dose, about 37 mg per dose, about 38 mg per dose, about 39 mg per dose, about 40 mg per dose, about 41 mg per dose, about 42 mg per dose, about 43 mg per dose, about 44 mg per dose, about 45 mg per dose, about 46 mg per dose, about 47 mg per dose, about 48 mg per dose, about 49 mg per dose, about 50 mg per dose, about 51 mg per dose, about 52 mg per dose, about 53 mg per dose, about 54 mg per dose, about 55 mg per dose, about 56 mg per dose, about 57 mg per dose, about 58 mg per dose, In a specific embodiment, the FGF-21 conjugates disclosed herein, e.g., the PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, are present in the formulations disclosed herein in an amount of about 10 mg per dose. In a specific embodiment, the FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, is present in the formulation disclosed herein in an amount of about 20 mg per dose. In some embodiments, the dose is a flat dose.

[0213] In some embodiments, the pharmaceutical formulation is formulated for subcutaneous administration.As discussed below, other pharmaceutical formulations disclosed herein can be administered via other routes.In some embodiments, the pharmaceutical formulation is formulated for subcutaneous administration, for example, by safety syringe.In some embodiments, the formulation is formulated for daily or weekly administration, for example, every 1, 2, 3, 4, 5, 6 days, every week, or every other week.In some embodiments, the formulation is an aqueous formulation.

[0214] In some aspects, the disclosure provides a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4; (ii) histidine at a concentration of between about 10 mM and about 50 mM; (iii) sucrose at a concentration of between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration of between about 10 μM and about 100 μM, wherein the pH of the formulation is between about 6.7 and about 7.5.

[0215] In some aspects, the disclosure provides a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6; (ii) histidine at a concentration of between about 10 mM and about 50 mM; (iii) sucrose at a concentration of between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration of between about 10 μM and about 100 μM, wherein the pH of the formulation is between about 6.7 and about 7.5.

[0216] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5, or SEQ ID NO: 4 or 6; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 μM, wherein the pharmaceutical formulation has a pH of about 7.1.

[0217] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5, or SEQ ID NO: 4 or 6; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 μM, wherein the pH is 7.1.

[0218] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5, or SEQ ID NO: 4 or 6; (ii) histidine at a concentration of about 20 mM; and (iii) sucrose at a concentration of about 600 mM, wherein the pharmaceutical formulation has a pH of about 7.0.

[0219] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5, or SEQ ID NO: 4 or 6; (ii) histidine at a concentration of 20 mM; and (iii) sucrose at a concentration of 600 mM, wherein the pH is 7.0.

[0220] In some specific aspects, the present disclosure provides a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, at a concentration of about 10 mg / mL; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM, wherein the pH is about 7.1. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulation disclosed herein is measured by Slope Spectroscopy, e.g., an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0221] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, at a concentration of about 20 mg / mL; (ii) histidine at a concentration of about 20 mM; (iii) sucrose at a concentration of about 600 mM; (iv) polysorbate 80 at a concentration of about 0.05% (w / v); and (v) DTPA at a concentration of about 50 uM, wherein the pharmaceutical formulation has a pH of about 7.1. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulation disclosed herein is measured by Slope Spectroscopy, e.g., an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0222] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, at a concentration of 10 mg / mL; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0223] Also provided is a pharmaceutical formulation comprising: (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, at a concentration of 20 mg / mL; (ii) histidine at a concentration of 20 mM; (iii) sucrose at a concentration of 600 mM; (iv) polysorbate 80 at a concentration of 0.05% (w / v); and (v) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0224] The present disclosure also provides pharmaceutical formulations prepared according to any of the methods of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or 5, or SEQ ID NO:4 or 6.

[0225] In a specific embodiment, the disclosure provides a pharmaceutical formulation comprising an FGF-21 conjugate of SEQ ID NO: 4 or 6 at a concentration of 20 g / L in 20 mM histidine, 600 mM sucrose, 50 uM DTPA, and 0.05% PS80, pH 7.1. In a specific embodiment, the disclosure provides a pharmaceutical formulation comprising an FGF-21 conjugate of SEQ ID NO: 4 or 6 at a concentration of 10 g / L in 20 mM histidine, 600 mM sucrose, 50 uM DTPA, and 0.05% PS80 (w / v), pH 7.1. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulation disclosed herein is measured by Slope Spectroscopy, e.g., an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0226] In some embodiments, the formulation is frozen. In some embodiments, the formulation is stored in a bag, e.g., a clamshell bag. In some embodiments, the bag, e.g., the clamshell bag, has a volume between 6L and 12L.

[0227] In some aspects, the formulation is contained in a vial. In some aspects, the formulation is contained in a syringe. In some aspects, the syringe is a safety syringe. In some aspects, the syringe is a prefillable syringe. In some aspects, the syringe is a BD NEOPAK™ prefillable syringe. In some aspects, the formulation is contained in an autoinjection device.

[0228] In some embodiments, the syringe (e.g., a pre-fillable syringe) or auto-injection device contains 1 mL of a pharmaceutical formulation comprising an FGF-21 conjugate of SEQ ID NO: 4 or 6 at a concentration of 10 g / L in 20 mM histidine, 600 mM sucrose, 50 uM DTPA, and 0.05% PS80 (w / v), pH 7.1. In some embodiments, the syringe (e.g., a pre-fillable syringe) or auto-injection device contains 1 mL of a pharmaceutical formulation comprising an FGF-21 conjugate of SEQ ID NO: 4 or 6 at a concentration of 20 g / L in 20 mM histidine, 600 mM sucrose, 50 uM DTPA, and 0.05% PS80, pH 7.1. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulation disclosed herein is measured by Slope Spectroscopy, e.g., an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm. II. Manufacturing method

[0229] The present disclosure also provides a method for improving or enhancing the stability of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, comprising the step of incorporating an aminopolycarboxylic acid cation chelator, such as DTPA, wherein the formulation has improved stability compared to a reference formulation that does not contain the aminopolycarboxylic acid cation chelator. These stabilization methods include (i) incorporating an aminopolycarboxylic acid cation chelator into the formulation, (ii) incorporating a surfactant that is a polysorbate, such as polysorbate 80, (iii) adjusting the pH of the formulation to about 7.1, or (iv) any combination thereof.

[0230] In some embodiments, the pharmaceutical formulations described herein are made by the process of mixing (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, in an amount to achieve a final concentration of about 10 mg / mL; (ii) histidine in an amount to achieve a final concentration of about 20 mM; (iii) sucrose in an amount to achieve a final concentration of about 600 mM; (iv) polysorbate 80 in an amount to achieve a final concentration of about 0.05% (w / v); and (v) DTPA in an amount to achieve a final concentration of about 50 uM; and adjusting the pH to about 7.1.

[0231] In some embodiments, the pharmaceutical formulations described herein are made by the process of mixing (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, in an amount to achieve a final concentration of about 20 mg / mL; (ii) histidine in an amount to achieve a final concentration of about 20 mM; (iii) sucrose in an amount to achieve a final concentration of about 600 mM; (iv) polysorbate 80 in an amount to achieve a final concentration of about 0.05% (w / v); and (v) DTPA in an amount to achieve a final concentration of about 50 uM; and adjusting the pH to about 7.1.

[0232] In some embodiments, the pharmaceutical formulations described herein are made by the process of mixing (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, in an amount to achieve a final concentration of 10 mg / mL; (ii) histidine in an amount to achieve a final concentration of 20 mM; (iii) sucrose in an amount to achieve a final concentration of 600 mM; (iv) polysorbate 80 in an amount to achieve a final concentration of 0.05% (w / v); and (v) DTPA in an amount to achieve a final concentration of 50 uM; and adjusting the pH to 7.1.

[0233] In some embodiments, the pharmaceutical formulations described herein are made by the process of mixing (i) an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or 5 or SEQ ID NO: 4 or 6, in an amount to achieve a final concentration of 20 mg / mL; (ii) histidine in an amount to achieve a final concentration of 20 mM; (iii) sucrose in an amount to achieve a final concentration of 600 mM; (iv) polysorbate 80 in an amount to achieve a final concentration of 0.05% (w / v); and (v) DTPA in an amount to achieve a final concentration of 50 uM; and adjusting the pH to 7.1.

[0234] As used herein, the term "mixing" refers to combining the components of the formulations disclosed herein in any manner known in the art in a non-predetermined order to reach the disclosed concentrations. For example, the excipients in the pharmaceutical formulations disclosed herein may be added sequentially or simultaneously to a solution containing the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4. Alternatively, a concentrated solution containing the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be added to a solution containing all or a portion of the excipients in the formulation. In other embodiments, the excipients may be incorporated into the formulation, for example, using dialysis or filtration.

[0235] In some aspects, the improved stability resulting from application of the disclosed methods includes, for example, (i) increased physical stability relative to a reference formulation, (ii) increased chemical stability relative to a reference formulation, or (iii) a combination thereof.

[0236] In some aspects, increasing physical stability comprises (i) preventing or reducing FGF-21 polypeptide aggregation, (ii) preventing or reducing FGF-21 polypeptide fragmentation, or (iii) a combination thereof.

[0237] In some aspects, the FGF-21 polypeptide aggregation observed in a pharmaceutical composition disclosed herein is about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% of the FGF-21 polypeptide aggregation observed in a reference formulation.

[0238] In some aspects, FGF-21 polypeptide aggregation observed in a pharmaceutical composition disclosed herein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the FGF-21 polypeptide aggregation observed in a reference formulation.

[0239] In some aspects, the FGF-21 polypeptide fragmentation observed in a pharmaceutical composition disclosed herein is about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% of the FGF-21 polypeptide fragmentation observed in a reference formulation.

[0240] In some aspects, the FGF-21 polypeptide fragmentation observed in a pharmaceutical composition disclosed herein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the FGF-21 polypeptide fragmentation observed in a reference formulation.

[0241] In some aspects, the increased chemical stability comprises (i) preventing or reducing deamidation of the FGF-21 polypeptide, (ii) preventing or reducing oxidation of the FGF-21 polypeptide, or (iii) a combination thereof. In some aspects, the deamidation of the FGF-21 polypeptide observed in the pharmaceutical compositions disclosed herein is about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% of the deamidation of the FGF-21 polypeptide observed in the reference formulation.

[0242] In some aspects, the deamidation of FGF-21 polypeptide observed in a pharmaceutical composition disclosed herein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the deamidation of FGF-21 polypeptide observed in a reference formulation.

[0243] In some aspects, the oxidation of FGF-21 polypeptide observed in a pharmaceutical composition disclosed herein is about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% of the oxidation of FGF-21 polypeptide observed in a reference formulation.

[0244] In some aspects, the oxidation of FGF-21 polypeptide observed in a pharmaceutical composition disclosed herein is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% of the oxidation of FGF-2 polypeptide observed in a reference formulation.

[0245] In some aspects, methods of stabilizing (improving or enhancing the stability of) the formulations disclosed herein may reduce not only the level of degradation but also the rate of degradation: for example, the disclosed methods may reduce the deamidation rate of the polypeptide, reduce oxidation of the FGF-21 polypeptide, reduce aggregation of the FGF-21 polypeptide, reduce proteolysis of the FGF-21 polypeptide, or any combination thereof, relative to a reference formulation.

[0246] For example, the incorporation of an aminopolycarboxylic acid cation chelator, such as DTPA, into a pharmaceutical formulation disclosed herein may reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, relative to a reference formulation when stored at a certain temperature for a certain period of time.

[0247] In some aspects, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the deamidation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored, e.g., at about 25°C, about 30°C, about 35°C, about 40°C, or about 45°C.

[0248] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) may reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, relative to a reference formulation when the pharmaceutical formulation is stored at a temperature above 25°C, above 30°C, above 35°C, about 40°C, or about 45°C.

[0249] In some embodiments, an aminopolycarboxylate cation chelator (e.g., DTPA) may reduce the deamidation rate of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, relative to a reference formulation when the pharmaceutical formulation is stored at between about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, or about 40°C to about 45°C.

[0250] In some embodiments, the incorporation of an aminopolycarboxylate cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the deamidation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at a temperature or temperature range disclosed above for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or about 4 months.

[0251] In a specific embodiment, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, can reduce the deamidation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at 40° C. for about one month.

[0252] In some aspects, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the high molecular weight (HMW) aggregation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at about 25°C, about 30°C, about 35°C, about 40°C, or about 45°C.

[0253] In some embodiments, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the HMW aggregation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at a temperature greater than 25°C, greater than 30°C, greater than 35°C, about 40°C, or about 45°C.

[0254] In some embodiments, the incorporation of an aminopolycarboxylate cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the HMW aggregation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at a temperature between about 20°C to about 25°C, about 25°C to about 30°C, about 30°C to about 35°C, or about 40°C to about 45°C.

[0255] In some embodiments, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may reduce the HMW aggregation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at a temperature or temperature range disclosed above for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, or about 4 months.

[0256] In a specific embodiment, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, can reduce the HMW aggregation rate of the FGF-21 conjugate relative to a reference formulation when the pharmaceutical formulation is stored at 40° C. for about one month.

[0257] In some aspects, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation containing an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, can prevent or mitigate oxidation of one or more methionines within the FGF-21 conjugate.

[0258] In certain aspects, the incorporation of an aminopolycarboxylic acid cation chelator (e.g., DTPA) into a pharmaceutical formulation containing an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, can prevent or mitigate the oxidation of amino acid 1 and / or amino acid 169 of SEQ ID NO:3 (wild-type FGF-21), or the corresponding amino acids in SEQ ID NOs:1, 2, 4, or any other FGF-21 conjugate according to this disclosure, e.g., at 25°C and / or 40°C.

[0259] The disclosed method may use DTPA, which is an aminopolycarboxylic acid cation chelator.However, in some other embodiments, the aminopolycarboxylic acid cation chelator may be a DTPA analogue, such as EDTA, EGTA, DOTA, tiuxetan, or any other aminopolycarboxylic acid cation chelator known in the art that is analogous to DTPA and EDTA, such as DTPA.BMA and EDTA.BMA.

[0260] In some embodiments, the DTPA cationic chelator may be mixed in an amount of between about 10 μM and about 100 μM, between 15 μM and about 95 μM, between about 20 μM and about 90 μM, between about 25 μM and about 85 μM, between about 30 μM and about 80 μM, between about 35 μM and about 75 μM, between about 40 μM and about 70 μM, between about 45 μM and about 65 μM, between about 50 μM and about 60 μM, between about 25 μM and about 75 μM, between about 40 μM and about 60 μM, between about 30 μM and about 70 μM, or between about 40 μM and about 75 μM.

[0261] In some embodiments, the DTPA cation chelator may be mixed in an amount of about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, about 50 μM, about 55 μM, about 60 μM, about 65 μM, about 70 μM, about 75 μM, about 80 μM, about 85 μM, about 90 μM, about 95 μM, or about 100 μM.

[0262] In some embodiments, the DTPA cation chelator may be admixed in an amount of at least about 15 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, at least about 55 μM, at least about 60 μM, at least about 65 μM, at least about 70 μM, or at least about 75 μM.

[0263] In a specific embodiment, the aminopolycarboxylate cation chelator, eg, DTPA, is admixed in an amount of 50 μM.

[0264] In some embodiments of the methods disclosed herein, the pH of the formulation is adjusted to a pH greater than about 6.5, greater than about 6.6, greater than about 6.7, greater than about 6.8, greater than about 6.9, greater than about 7.0, greater than about 7.1, greater than about 7.2, greater than about 7.3, greater than about 7.4, or greater than about 7.5.

[0265] In some embodiments, the pH of the formulation is adjusted to a pH greater than 6.5, greater than 6.6, greater than 6.7, greater than 6.8, greater than 6.9, greater than 7.0, greater than 7.1, greater than 7.2, greater than 7.3, greater than 7.4, or greater than 7.5.

[0266] In some embodiments, the pH of the formulation is adjusted to a pH of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0267] In some embodiments, the pH of the formulation is between about 6.5 to about 7.5, about 6.6 to about 7.5, about 6.7 to about 7.5, about 6.8 to about 7.5, about 6.9 to about 7.5, about 7.0 to about 7.5, about 7.1 to about 7.5, about 7.2 to about 7.5, about 7.3 to about 7.5, about 7.4 to about 7.5, about 6.5 to about 7.4, about 6.5 to about 7.3, about 6.5 to about 7.2, about 6.5 to about 7.1, about 6.5 to about 7.0, about 6.5 to about 6.9, about 6.5 to about 6.8, about 6.5 to about 6.7, about 6.6 to about 7.4, about 6.7 to about 7.4, about 6.8 to about 7.4, about 6.9 to about 7. 4, about 7.0 to about 7.4, about 7.1 to about 7.4, about 7.2 to about 7.4, about 7.3 to about 7.4, about 6.5 to about 7.3, about 6.6 to about 7.3, about 6.7 to about 7.3, about 6.7 to about 7.3, about 6.8 to about 7.3, about 6.9 to about 7.3, about 7.0 to about 7.3, about 7.1 to about 7.3, about 7.2 to about 7.3, about 6.5 to about 7.2, about 6.6 to about 7.2, about 6.7 to about 7.2, about 6.8 to about 7.2, about 6.9 to about 7.2, about 7.0 to about 7.2, about 7.1 to about 7.2, about 6.9 to about 7.1, or about 7.0 to about 7.1.

[0268] In some embodiments, the pH of the formulation is adjusted to a pH of about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the pharmaceutical formulation is more stable after the pH is adjusted than a reference formulation having a pH of 6.5.

[0269] In some embodiments, the pH of the formulation is adjusted to a pH of 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some embodiments, the pharmaceutical formulation is more stable after the pH is adjusted than a reference formulation having a pH of 6.5.

[0270] In some embodiments, the method for improving the stability of pharmaceutical formulations comprising FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, further comprises mixing a surfactant. In some embodiments, the mixed surfactant is a non-ionic surfactant, i.e., a surfactant that tends to have no net charge in neutral solution. In some embodiments, the mixed non-anionic surfactant is a polysorbate. In some embodiments, the non-ionic surfactant is mixed in an amount above the critical micelle concentration (CMC), which is at least about 0.01 mg / ml for polyoxyethylene sorbitan fatty acid esters. See Wan and Lee, Journal of Pharm Sci, 63, p.136, 1974. In some embodiments of the method, the polysorbate is polysorbate 80 (PS80).

[0271] In some embodiments, the surfactant that is PS80 is about 0.01% to about 0.1% (w / v), about 0.02% to about 0.1% (w / v), about 0.03% to about 0.1% (w / v), about 0.04% to about 0.1% (w / v), about 0.05% to about 0.1% (w / v), about 0.06% to about 0.1% (w / v), about 0.07% to about 0.1% (w / v), about 0.08% to about 0.1% (w / v), about 0.09% to about 0.1% (w / v), about 0.02% to about 0.09% (w / v), about 0.03% to about 0.09% (w / v), about 0.04% to about 0.09% (w / v), about 0.05% to about 0.09 ... % (w / v), about 0.06% to about 0.09% (w / v), about 0.07% to about 0.09% (w / v), about 0.08% to about 0.09% (w / v), about 0.03% to about 0.08% (w / v), about 0.04% to about 0.08% (w / v), about 0.05% to about 0.08% (w / v), about 0.06% to about 0.08% (w / v), about 0.07% to about 0.08% (w / v), about 0.04% to about 0.07% (w / v), about 0.05% to about 0.07% (w / v), about 0.06% to about 0.07% (w / v), or about 0.05% to about 0.06% (w / v).

[0272] In some embodiments, the surfactant, polysorbate 80, is mixed in an amount of at least about 0.01% (w / v), at least about 0.02% (w / v), at least about 0.03% (w / v), at least about 0.04% (w / v), at least about 0.05% (w / v), at least about 0.06% (w / v), at least about 0.07% (w / v), at least about 0.08% (w / v), at least about 0.09% (w / v), or at least about 0.1% (w / v). In some embodiments, the surfactant, e.g., PS80, is mixed in an amount sufficient to mitigate or prevent particulate formation and / or air bubble formation, e.g., when the formulation is agitated (e.g., on a shaker).

[0273] In some embodiments, the method of improving the stability of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, further comprises admixing an amino acid buffer, e.g., histidine (i.e., L-histidine, D-histidine, solvated histidine, hydrated histidine, anhydrous histidine, or a mixture thereof).

[0274] In some embodiments, the histidine buffer is about 10 mM to about 100 mM histidine, about 15 mM to about 100 mM histidine, about 20 mM to about 100 mM histidine, about 25 mM to about 100 mM histidine, about 30 mM to about 100 mM histidine, about 35 mM to about 100 mM histidine, about 40 mM to about 100 mM histidine, about 45 mM to about 100 mM histidine, about 50 mM to about 100 mM histidine, about 55 mM to about 100 mM histidine, about 60 mM to about 100 mM histidine, about 65 mM to about 100 mM histidine, about 70 mM to about 100 mM histidine. , about 75 mM to about 100 mM histidine, about 80 mM to about 100 mM histidine, about 85 mM to about 100 mM histidine, about 90 mM to about 100 mM histidine, about 95 mM to about 100 mM histidine, about 20 mM to about 90 mM histidine, about 25 mM to about 90 mM histidine, about 30 mM to about 90 mM histidine, about 35 mM to about 90 mM histidine, about 40 mM to about 90 mM histidine, about 45 mM to about 90 mM histidine, about 50 mM to about 90 mM histidine, about 55 mM to about 90 mM histidine, about 60 mM to about 90 mM histidine, about 65 mM about 90 mM histidine, about 70 mM to about 90 mM histidine, about 75 mM to about 90 mM histidine, about 80 mM to about 90 mM histidine, about 85 mM to about 90 mM histidine, about 30 mM to about 80 mM histidine, about 35 mM to about 80 mM histidine, about 40 mM to about 80 mM histidine, about 45 mM to about 80 mM histidine, about 50 mM to about 80 mM histidine, about 55 mM to about 80 mM histidine, about 60 mM to about 80 mM histidine, about 65 mM to about 80 mM histidine, about 70 mM to about 80 mM histidine, about 75 mM to about 80 mM histidine, about 40 mM to about 70 mM histidine, about 45 mM to about 70 mM histidine, about 50 mM to about 70 mM histidine, about 55 mM to about 70 mM histidine, about 60 mM to about 70 mM histidine, about 65 mM to about 70 mM histidine, about 10 mM to about 30 mM histidine, about 15 mM to about 30 mM histidine, about 20 mM to about 30 mM histidine, about 25 mM to about 30 mM histidine, about 15 mM to about 25 mM histidine, about 20 mM to about 25 mM, about 15 mM to about 20 mM histidine, about 40 mM to about 60 mM histidine, about 45 mM to about 60 mM histidine,It is mixed in an amount of about 50 mM to about 60 mM histidine, about 15.5 mM to about 24.5 mM histidine, about 16 mM to about 24 mM histidine, about 16.5 mM to about 23.5 mM histidine, about 17 mM to about 23 mM histidine, about 17.5 mM to about 22.5 mM histidine, about 18 mM to about 22 mM histidine, about 18.5 mM to about 21.5 mM histidine, about 19 mM to about 21 mM histidine, or about 19.5 mM to about 20.5 mM histidine.

[0275] In some embodiments, the histidine buffer is about 10 mM histidine, about 11 mM histidine, about 12 mM histidine, about 13 mM histidine, about 14 mM histidine, about 15 mM histidine, about 16 mM histidine, about 17 mM histidine, about 18 mM histidine, about 19 mM histidine, about 20 mM histidine, about 21 mM histidine, about 22 mM histidine, about 23 mM histidine, about 24 mM histidine, about 25 mM histidine, about 26 mM histidine, about 27 mM histidine, about 28 mM histidine, about 29 mM histidine, about 30 mM histidine, about 31 mM histidine, about 32 mM histidine, about 33 mM histidine, about 34 mM histidine, about 35 mM histidine, about 36 mM histidine, about 37 mM histidine, about 38 mM histidine, about 39 mM histidine, about 40 mM histidine, about 41 mM histidine, about 42 mM histidine, about 43 mM histidine, about 44 mM histidine, about 45 mM histidine, about 46 mM histidine, about 47 mM histidine, about 48 mM histidine, about 49 mM histidine, about 50 mM histidine, about 51 mM histidine, about 52 mM histidine, about 53 mM histidine, about 54 mM histidine, about 55 mM histidine, about 56 mM histidine, about 57 mM histidine, about 58 mM histidine, about 59 mM histidine, about 60 mM histidine, about 61 mM histidine, about 62 mM histidine, about 63 mM histidine, about 64 mM histidine, about 65 mM his histidine, about 31 mM histidine, about 32 mM histidine, about 33 mM histidine, about 34 mM histidine, about 35 mM histidine, about 36 mM histidine, about 37 mM histidine, about 38 mM histidine, about 39 mM histidine, about 40 mM histidine, about 41 mM histidine, about 42 mM histidine, about 43 mM histidine, about 44 mM histidine, about 45 mM histidine, about 46 mM histidine, about 47 mM histidine, about 48 mM histidine, about 49 mM histidine, or about 50 mM histidine.

[0276] In some embodiments, the method of improving the stability of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, further comprises mixing an osmotic agent (isotonicity agent). In accordance with the present disclosure, the osmotic agent (isotonicity agent) may comprise a polyol, a sugar, a carbohydrate, a salt such as sodium chloride, or a mixture thereof. Exemplary polyols include polyols with a molecular weight of less than about 600 kD (e.g., in the range of 120-400 kD), e.g., mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or a mixture thereof. Osmotic agents that are sugars or carbohydrates include monosaccharides, disaccharides, and polysaccharides, or a mixture thereof.

[0277] In some aspects, the sugar or carbohydrate is selected from the group consisting of fructose, glucose, mannose, sucrose, sorbose, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan, and mixtures thereof. In some aspects, the osmotic agent comprises a sugar selected from the group of reducing or non-reducing sugars, or mixtures thereof. In some aspects, the osmotic agent or tonicity agent comprises a sugar that is a non-reducing sugar, preferably selected from the group consisting of sucrose, trehalose, or mixtures thereof. In some specific aspects, the non-reducing sugar is sucrose.

[0278] In some embodiments, the osmotic regulator that is sucrose is about 100 mM to about 1 M sucrose, about 200 mM to about 1 M sucrose, about 300 mM to about 1 M sucrose, about 400 mM to about 1 M sucrose, about 500 mM to about 1 M sucrose, about 600 mM to about 1 M sucrose, about 700 mM to about 1 M sucrose, about 800 mM to about 1 M sucrose, about 900 mM to about 1 M sucrose, about 200 mM to about 900 mM sucrose, about 300 mM to about 900 mM sucrose, about 400 mM to about 900 mM sucrose, about 500 mM to about 900 mM sucrose. sucrose, about 600 mM to about 900 mM sucrose, about 700 mM to about 900 mM sucrose, about 800 mM to about 900 mM sucrose, about 300 mM to about 800 mM sucrose, about 400 mM to about 800 mM sucrose, about 500 mM to about 800 mM sucrose, about 600 mM to about 800 mM sucrose, about 700 mM to about 800 mM sucrose, about 400 mM to about 700 mM sucrose, about 500 mM to about 700 mM sucrose, about 600 mM to about 700 mM sucrose, or about 500 mM to about 600 mM sucrose.

[0279] In some embodiments, the osmotic modifier that is sucrose is mixed in an amount of about 100 mM sucrose, about 150 mM sucrose, about 200 mM sucrose, about 250 mM sucrose, about 300 mM sucrose, about 350 mM sucrose, about 400 mM sucrose, about 450 mM sucrose, about 500 mM sucrose, about 550 mM sucrose, about 600 mM sucrose, about 650 mM sucrose, about 700 mM sucrose, about 750 mM sucrose, about 800 mM sucrose, about 850 mM sucrose, about 900 mM sucrose, about 950 mM sucrose, or about 1 M sucrose.

[0280] In some aspects, an FGF-21 conjugate useful in the methods disclosed herein comprises an FGF-21 polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:3 (wild-type human FGF-21), wherein the FGF-21 polypeptide has FGF-21 activity.

[0281] In some embodiments, the FGF-21 conjugate is PEG-FGF-21 of SEQ ID NO: 2, i.e., FGF-21 of SEQ ID NO: 1, in which glutamine 109 (SEQ ID NO: 3) of wild-type FGF-21 is replaced with para-acetyl-L-phenylalanine and a PEG moiety, e.g., a linear PEG of molecular weight between about 28 kDa and about 32 kDa, e.g., about 30 kDa, is covalently attached to the para-acetyl-L-phenylalanine via an oxime linkage. In some embodiments, the FGF-21 conjugate is PEG-FGF-21 of SEQ ID NO: 4, i.e., FGF-21 of SEQ ID NO: 1, in which glutamine 109 (SEQ ID NO: 3) of wild-type FGF-21 is replaced with para-acetyl-L-phenylalanine and a PEG moiety comprising 681 ethylene glycol units is covalently attached to the para-acetyl-L-phenylalanine via an oxime linkage.

[0282] In some embodiments of the methods disclosed herein, the FGF-21 conjugate, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is present at a concentration between about 1 mg / ml and about 40 mg / ml. In some embodiments, the FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is present at a concentration of about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, or about 40 mg / ml. In some specific embodiments, the FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is present at a concentration of about 10 mg / ml. In some specific embodiments, the FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is present at a concentration of about 20 mg / ml.

[0283] In some embodiments, the formulation comprises about 1 mL of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 10 mg / ml. In some embodiments, the formulation comprises about 1 mL of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 20 mg / ml.

[0284] In some embodiments, the concentration of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, is determined according to methods known in the art or specific methods disclosed in the Examples section herein.

[0285] In some aspects of the methods disclosed herein, the formulation is formulated for subcutaneous administration. In some aspects, the formulation is formulated for subcutaneous administration with a safety syringe. In some aspects, the formulation is formulated for daily or weekly administration. In some aspects, the formulation is an aqueous formulation.

[0286] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising the steps of mixing: (i) histidine at a concentration of between about 10 mM and about 50 mM; (ii) sucrose at a concentration of between about 100 mM and about 1 M; (iii) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (iv) DTPA at a concentration of between about 10 μM and about 100 μM, wherein the pH of the formulation is between about 6.7 and about 7.5.

[0287] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising the steps of mixing: (i) histidine at a concentration of between about 15 mM and about 45 mM; (ii) sucrose at a concentration of between about 200 mM and about 900 mM; (iii) polysorbate 80 at a concentration of between about 0.02% and about 0.09% (w / v); and (iv) DTPA at a concentration of between about 20 μM and about 90 μM, wherein the pH of the formulation is between about 6.8 and about 7.4.

[0288] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising the steps of mixing: (i) histidine at a concentration of between about 15 mM and about 40 mM; (ii) sucrose at a concentration of between about 300 mM and about 800 mM; (iii) polysorbate 80 at a concentration of between about 0.03% and about 0.08% (w / v); and (iv) DTPA at a concentration of between about 30 μM and about 80 μM, wherein the pH of the formulation is between about 6.9 and about 7.3.

[0289] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising the steps of mixing: (i) histidine at a concentration of between about 15 mM and about 30 mM; (ii) sucrose at a concentration of between about 400 mM and about 800 mM; (iii) polysorbate 80 at a concentration of between about 0.04% and about 0.07% (w / v); and (iv) DTPA at a concentration of between about 40 μM and about 70 μM, wherein the pH of the formulation is between about 7 and about 7.2.

[0290] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising the steps of mixing: (i) histidine at a concentration of between about 15 mM and about 25 mM; (ii) sucrose at a concentration of between about 500 mM and about 700 mM; (iii) polysorbate 80 at a concentration of between about 0.04% and about 0.06% (w / v); and (iv) DTPA at a concentration of between about 45 μM and about 55 μM, wherein the pH of the formulation is between about 7 and about 7.1. In some embodiments, the concentration of FGF-21 conjugate in the pharmaceutical formulation disclosed herein is measured by Slope Spectroscopy, e.g., an Agilent Cary 60 UV-Vis spectrometer equipped with a SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm.

[0291] The present disclosure also provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 μM, wherein the pH is about 7.1.

[0292] The present disclosure also provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 μM, wherein the pH is 7.1.

[0293] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing: (i) histidine at a concentration of about 20 mM; and (ii) sucrose at a concentration of about 600 mM, wherein the pH is about 7.0.

[0294] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing: (i) histidine at a concentration of 20 mM; and (ii) sucrose at a concentration of 600 mM, wherein the pH is 7.0.

[0295] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 10 mg / mL, comprising mixing: (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 uM, wherein the pH is about 7.1.

[0296] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 20 mg / mL, comprising mixing: (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 uM, wherein the pH is about 7.1.

[0297] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 10 mg / mL, comprising mixing: (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0298] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 20 mg / mL, comprising mixing (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0299] The present disclosure also provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 μM, wherein the pH is about 7.1.

[0300] The present disclosure also provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 μM, wherein the pH is 7.1.

[0301] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing: (i) histidine at a concentration of about 20 mM; and (ii) sucrose at a concentration of about 600 mM, wherein the pH is about 7.0.

[0302] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6 (e.g., at a concentration of 10 mg / mL or 20 mg / mL), comprising mixing: (i) histidine at a concentration of 20 mM; and (ii) sucrose at a concentration of 600 mM, wherein the pH is 7.0.

[0303] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6, at a concentration of about 10 mg / mL, comprising mixing: (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 uM, wherein the pH is about 7.1.

[0304] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6, at a concentration of about 20 mg / mL, comprising mixing: (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; (iii) polysorbate 80 at a concentration of about 0.05% (w / v); and (iv) DTPA at a concentration of about 50 uM, wherein the pH is about 7.1.

[0305] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6, at a concentration of about 10 mg / mL, comprising mixing (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0306] The present disclosure provides a method of improving the stability of a formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6, at a concentration of about 20 mg / mL, comprising mixing (i) histidine at a concentration of 20 mM; (ii) sucrose at a concentration of 600 mM; (iii) polysorbate 80 at a concentration of 0.05% (w / v); and (iv) DTPA at a concentration of 50 uM, wherein the pH is 7.1.

[0307] In some embodiments, the concentration of an individual excipient (e.g., DTPA, PS80, histidine, sucrose, or combinations thereof) included in a pharmaceutical formulation may be determined / calculated to be the amount (e.g., weight, moles, etc.) of the individual excipient added to the pharmaceutical formulation during its manufacture per final volume unit of the finished pharmaceutical formulation. In other embodiments, the concentration of an excipient (e.g., DTPA, PS80, histidine, sucrose, or combinations thereof) included in a pharmaceutical formulation is based on the actual amount of the individual excipient in the pharmaceutical formulation.

[0308] In some embodiments, the manufacturing methods disclosed herein further comprise transferring a formulation disclosed herein, e.g., a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 10 mg or about 20 mg / mL, histidine at a concentration of 20 mM, sucrose at a concentration of 600 mM, polysorbate 80 at a concentration of 0.05% (w / v), and DTPA at a concentration of 50 uM, wherein the pH is 7.1, into a container. In some embodiments, the manufacturing method disclosed herein further comprises transferring a formulation disclosed herein, e.g., a formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 5 or SEQ ID NO: 6, at a concentration of about 10 mg or about 20 mg / mL, histidine at a concentration of 20 mM, sucrose at a concentration of 600 mM, polysorbate 80 at a concentration of 0.05% (w / v), and DTPA at a concentration of 50 uM, wherein the pH is 7.1, to a container. In some embodiments, the container is a vial. In some embodiments, the container is a bag, e.g., a clamshell bag. In some embodiments, the bag, e.g., a clamshell bag, has a volume between 6 L and 12 L. In some embodiments, the container is a syringe. In some embodiments, the syringe is a safety syringe. In some embodiments, the syringe is a pre-fillable syringe. In some embodiments, the syringe is a BD NEOPAK™ prefillable syringe. In some embodiments, the container is an autoinjection device. In some embodiments, about 1 mL of a formulation containing an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, at a concentration of about 10 mg or about 20 mg / mL, is deposited in the container (e.g., a vial, a syringe, or an autoinjection device). In some embodiments, about 1 mL of a formulation containing an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 5 or SEQ ID NO: 6, at a concentration of about 10 mg or about 20 mg / mL, is deposited in the container (e.g., a vial, a syringe, or an autoinjection device). In some embodiments, the container contains multiple doses, for example, multiple 1 mL doses. III. Treatment

[0309] The present disclosure also provides a method of treating or preventing a disease or condition associated with fibrosis and / or diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0310] In some embodiments, the disease or condition is diabetes, e.g., diabetes type 2. In some embodiments, the disease or condition is non-alcoholic steatohepatitis (NASH).

[0311] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be used in the treatment or prevention methods disclosed herein in an amount between about 1 mg and about 40 mg per dose (e.g., a 1 mL dose or multiples thereof). In some embodiments, the amount of FGF-21 conjugate per dose is based on measurements by Slope Spectroscopy, e.g., Agilent Cary 60 UV-Vis Spectrometer equipped with SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE Disposable UV Plastic Vessel (C Technologies, Inc.; P / NOC0009-C1)), using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm. In some embodiments, the FGF-21 conjugates disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be used in the treatment or prevention methods disclosed herein in an amount between about 1 mg and about 5 mg per dose, or between about 1 mg and about 10 mg per dose, or between about 1 mg and about 15 mg per dose, or between about 1 mg and about 20 mg per dose, or between about 1 mg and about 25 mg per dose, or between about 1 mg and about 30 mg per dose, or between about 1 mg and about 35 mg per dose, or between about 1 mg and about 40 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In some embodiments, the FGF-21 conjugates disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be used in the treatment or prevention methods disclosed herein in an amount greater than 40 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In some embodiments, the dose is a flat dose.

[0312] In some embodiments, the FGF-21 conjugates disclosed herein, e.g., the PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be used in the treatment or prevention methods disclosed herein in an amount of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, or about 40 mg per dose. In a specific embodiment, the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, can be used in the treatment or prevention methods disclosed herein in an amount of about 10 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In a specific embodiment, the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, can be used in the treatment or prevention methods disclosed herein in an amount of about 20 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In some embodiments, the dose is a flat dose.

[0313] In some aspects, the pharmaceutical formulation is administered subcutaneously, e.g., using a safety syringe or an autoinjector. In some aspects, the pharmaceutical formulation is administered daily or weekly.

[0314] In some embodiments, administration of a pharmaceutical formulation comprising an effective amount of an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, to a subject reduces liver stiffness, reduces body fat percentage, reduces body weight, reduces liver weight to body weight ratio, reduces liver lipid content, reduces liver fibrosis area, reduces fasting blood glucose levels, reduces fasting triglyceride levels, reduces LDL cholesterol levels, reduces ApoB levels, reduces ApoC levels, increases HDL cholesterol, or any combination thereof.

[0315] In some embodiments, administration of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, to a subject in accordance with the treatment methods disclosed herein results in: (i) a reduction in liver fat levels; (ii) a reduction in liver damage levels; (iii) a reduction in fibrosis levels; (iv) a reduction in serum Pro-C3 (N-terminal collagen type III propeptide), a biomarker of fibrosis; (v) a reduction in alanine aminotransferase (ALT) levels; (vi) a reduction in aspartate aminotransferase (AST) levels; (vii) an increase in serum adiponectin levels; (viii) a reduction in plasma LDL levels; (ix) an increase in plasma HDL levels; (x) a reduction in plasma triglyceride levels; (xi) a reduction in liver stiffness levels; or (xii) any combination thereof, compared to levels in an untreated subject or in the subject prior to administration of the pharmaceutical formulation.

[0316] The present disclosure provides a method of treating a disease associated with fibrosis, comprising administering to a subject in need thereof an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0317] In some embodiments, the disease associated with fibrosis may affect organs or tissues such as the pancreas, lungs, heart, kidneys, liver, eyes, nervous system, bone marrow, lymph nodes, endocardium, and / or retroperitoneum. In some embodiments, the disease associated with fibrosis may be liver fibrosis or pre-cirrhosis. In some embodiments, the disease associated with fibrosis is non-alcoholic steatohepatitis (NASH), liver cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, pulmonary fibrosis, progressive massive fibrosis, idiopathic pulmonary fibrosis, injection site fibrosis, renal fibrosis, chronic kidney disease, diabetic kidney disease, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myofibrosis, heart failure, acute heart failure, chronic heart failure, metabolic heart failure, cardiac fibrosis, cataractous fibrosis, cataract, ocular scarring, pancreatic fibrosis, skin fibrosis, intestinal fibrosis, intestinal stenosis, endomyocardial fibrosis, atrial fibrosis, mediastinal fibrosis, Crohn's disease, retroperitoneal fibrosis, keloids, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, arthrofibrosis, Peyronie's disease, The fibrosis may be selected from the group consisting of fibrosis, ... In some embodiments, the disease associated with fibrosis is caused by one or more of the following: old age, heart attack, stroke, myocardial injury, or left ventricular dysfunction.In some embodiments, the disease associated with fibrosis can be selected from renal fibrosis, glomerulonephritis, chronic kidney disease, chronic renal failure, and nephritis associated with systemic lupus erythematosus, cancer, physical obstruction, toxins, metabolic disease, immune disease, or diabetic nephropathy.In some embodiments, the disease associated with fibrosis is caused by one or more of the following: trauma, spinal cord injury, infection, surgery, ischemic injury, heart attack, burns, exposure to environmental pollutants, pneumonia, tuberculosis, or acute respiratory distress syndrome.In some aspects, the disease associated with fibrosis may be selected from pulmonary fibrosis, interstitial lung disease, human fibrotic lung disease, idiopathic pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, myocardial fibrosis, macular degeneration, renal retinopathy, vitreoretinopathy, Graves' ophthalmopathy, drug-induced ergotine poisoning, cardiovascular disease, atherosclerosis / restenosis, keloids and hypertrophic scars, primary or idiopathic myofibrosis, inflammatory bowel disease, collagenous colitis, ocular scarring and cataract fibrosis. In some aspects, the disease associated with fibrosis may be selected from NASH, hepatic fibrosis, and liver cirrhosis. In some aspects, the disease associated with fibrosis may be NASH. In some aspects, the disease associated with fibrosis may be selected from diabetic kidney disease, chronic kidney disease, and renal fibrosis. In some aspects, the disease associated with fibrosis may be selected from metabolic heart failure and cardiac fibrosis. In some aspects, the disease associated with fibrosis can be pulmonary fibrosis.

[0318] In some aspects, the present disclosure provides a method of reducing hepatic fat fraction in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, optionally wherein the subject is at risk of developing or has been diagnosed as having NASH.

[0319] In some aspects, the present disclosure provides a method of reducing liver stiffness, reducing body fat percentage, reducing body weight, reducing liver weight to body weight ratio, reducing liver lipid content, reducing liver fibrosis area, lowering fasting blood glucose levels, lowering fasting triglyceride levels, lowering LDL cholesterol levels, lowering ApoB levels, lowering ApoC levels, and / or increasing HDL cholesterol in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, optionally wherein the subject is at risk of developing or has been diagnosed as having NASH.

[0320] In some aspects, the present disclosure provides a method of increasing adiponectin levels in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, optionally wherein the subject is at risk of developing or has been diagnosed as having NASH.

[0321] In some aspects, the present disclosure provides a method of treating one or more symptoms associated with NASH in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0322] Also provided herein is a method of treating or preventing NASH in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation disclosed herein comprising a mutant FGF-21 polypeptide comprising SEQ ID NO:1, wherein the non-natural p-acetyl-phenylalanine residue is linked via an oxime linkage to a PEG moiety of about 28 kDa to about 32 kDa molecular weight. In some embodiments, the non-natural p-acetyl-phenylalanine replaces glutamine 109 (SEQ ID NO:3) of wild-type FGF-21. In some embodiments, the PEG moiety has a molecular weight of about 30 kDa. In some embodiments, the PEG moiety has between about 600 and about 800 ethylene glycol units. In some embodiments, the PEG moiety is a PEG moiety of about 28 kDa to about 32 kDa molecular weight. 681 It is.

[0323] In some specific aspects, the present disclosure provides a method of treating or preventing NASH in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation disclosed herein comprising an FGF-21 conjugate of SEQ ID NO:2 or SEQ ID NO:4.

[0324] In some embodiments, the subject may optionally exhibit fibrosis stage 1-3 according to NASH CRN, as determined by liver biopsy. In some embodiments, before treatment, the subject may exhibit a fatty liver index of at least about 60. In some embodiments, before treatment, the subject may optionally exhibit a liver fat fraction percentage of at least 10%, as determined by magnetic resonance imaging.

[0325] In some aspects, the disclosure provides a method of treating type 1 or type 2 diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0326] In some aspects, the present disclosure provides a method of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0327] In some aspects, the present disclosure provides a method of regulating at least one of glucose and lipid homeostasis, glucose uptake, GLUT1 expression, and / or serum concentrations of glucose, triglycerides, insulin, or glucagon in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4.

[0328] In some aspects, the present disclosure provides a method of increasing insulin sensitivity, increasing adiponectin levels, reducing blood glucose levels, reducing glucagon levels, reducing triglyceride levels, reducing fructosamine levels, reducing low-density cholesterol levels, or reducing C-reactive protein levels in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4.

[0329] In some aspects, the present disclosure provides a method of treating a condition or disorder selected from obesity, diabetes, pancreatitis, insulin resistance, hyperinsulinemia, impaired glucose tolerance, hyperglycemia, metabolic syndrome, impaired glucose tolerance, impaired glucose clearance, high blood glucose, and Prader-Willi syndrome in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4.

[0330] In some aspects, the present disclosure provides a method of treating an insulin-related condition or disorder selected from type A insulin resistance, type C insulin resistance (also known as HAIR-AN syndrome), Rabson-Mendenhall syndrome, Donohue syndrome, or elfin syndrome, hyperandrogenism, hirsutism, or acanthosis nigricans in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4.

[0331] In some embodiments, pharmaceutical formulations comprising the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be administered via injection. In some embodiments, pharmaceutical formulations comprising the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be administered via subcutaneous, IV, intraperitoneal, or intramuscular injection, for example, using a syringe (such as a safety syringe) or an autoinjector.

[0332] In some embodiments, pharmaceutical formulations comprising FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once every day, or less than about once every day. In some embodiments, pharmaceutical formulations comprising FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about twice every week, or less than about twice every week. In some embodiments, pharmaceutical formulations comprising FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once every week, or less than about twice every week. In some embodiments, pharmaceutical formulations comprising FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once every two weeks, or less than about twice every week.

[0333] In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once every three weeks, or less than about twice weekly.

[0334] In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once per month, or less than about once per month.

[0335] In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered as frequently as once every four weeks.

[0336] In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once daily.

[0337] In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered at a frequency of about once weekly.

[0338] In some embodiments, pharmaceutical formulations comprising the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered in an amount selected from about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95, and about 100 mg of the FGF-21 conjugates disclosed herein per dose. In a specific embodiment, pharmaceutical formulations comprising the FGF-21 conjugates disclosed herein, such as PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered in an amount of about 10 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In a specific embodiment, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, can be administered in an amount of about 20 mg per dose (e.g., a dose of 1 mL or a multiple thereof). In some embodiments, the dose is a flat dose.

[0339] For example, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4 herein, may be administered to a subject at a concentration of between about 0.1-100 mg per kg of recipient subject body weight. In some embodiments, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, may be administered to a subject at a concentration of between about 0.5-5 mg of FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, per kg of recipient subject body weight. In another embodiment, a pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, may be administered to a recipient subject at a frequency between once daily to once every two weeks, such as about once or twice weekly, once every two days, once every three days, once every four days, once every five days, or once every six days.

[0340] The pharmaceutical formulation of the present disclosure, i.e., the formulation comprising the FGF-21 conjugate disclosed herein, for example, PEG-FGF-21 of SEQ ID NO: 2 or SEQ ID NO: 4, can be administered by any conventional route suitable for proteins or peptides, including, but not limited to, parenteral route, for example, injection, including but not limited to subcutaneous injection or intravenous injection, or any other form of injection or infusion.In some embodiments, the pharmaceutical formulation can be administered using a syringe, for example, a safety syringe.In some embodiments, the pharmaceutical formulation can be administered using an autoinjector. IV. Manufactured Articles and Kits

[0341] The present disclosure also provides an article of manufacture or kit comprising (i) an FGF-21 pharmaceutical formulation comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, and (ii) instructions for use. In some embodiments, the article of manufacture or kit may comprise a container. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In some embodiments, the container may be formed from a variety of materials, such as glass, plastic, or metal. In some embodiments, the container holds a pharmaceutical formulation, e.g., a liquid formulation, comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, the container holds a pharmaceutical formulation, e.g., a liquid formulation, comprising an FGF-21 conjugate disclosed herein, e.g., PEG-FGF-21 of SEQ ID NO:5 or SEQ ID NO:6.

[0342] The label on the container or the label associated with the container may indicate instructions for reconstitution and / or use.For example, the label may indicate that the pharmaceutical formulation comprising the FGF-21 conjugate disclosed herein, for example, the PEG-FGF-21 of SEQ ID NO:2 or SEQ ID NO:4, should be diluted to the protein concentration described above.The label may further indicate that the formulation is a subcutaneous formulation that is useful or intended for subcutaneous administration.

[0343] In some embodiments, the container holding the formulation is a multi-use vial, for example, allowing for multiple administrations (e.g., 2-6 or more administrations) of the subcutaneous formulation. Alternatively, the container can be, for example, a pre-filled syringe containing the subcutaneous formulation.

[0344] The article of manufacture or kit may further comprise a second container, for example, containing a solvent. The article of manufacture may further comprise other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit or article of manufacture comprises a syringe (e.g., a safety syringe). In some embodiments, the kit or article of manufacture comprises an autoinjector.

[0345] It should be appreciated that the "Description of the Invention" section, and not the "Summary" and "Abstract" sections, are intended to be used to interpret the claims. The "Summary" and "Abstract" sections may set forth one or more exemplary aspects of the disclosure contemplated by the inventor(s), but may not set forth all of them, and thus are not intended to limit the scope of the disclosure and the appended claims in any way.

[0346] The disclosure has been described above with the aid of functional components illustrating the implementation of the specified functions and relationships. In this specification, the boundaries of these functional components have been arbitrarily defined for convenience of description. Alternative boundaries may be defined so long as the specified functions and relationships are appropriately implemented.

[0347] The foregoing description of specific embodiments will fully reveal the general nature of the disclosure, such that other practitioners may easily modify and / or adapt such specific embodiments for various applications by applying knowledge within the skill of the art, without departing from the concept of the disclosure and without undue experimentation. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology used herein is intended to be descriptive and not limiting, as the terminology or terminology used herein would be interpreted by those skilled in the art in light of the teaching and guidance.

[0348] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0349] The contents of all references cited (including literature references, patents, patent applications, and websites) that may be cited throughout this application, as well as the references cited therein, are expressly incorporated herein by reference in their entirety for any purpose. EXAMPLES

[0350] Example 1 Development of PEG-FGF-21 formulation For initial studies, PEG-FGF-21 was formulated at 7.5 mg / mL in 20 mM Tris, 250 mM sucrose, pH 8.3. The high deamidation rate observed in this formulation necessitated storage of the drug product frozen at -20° C. Therefore, further studies were conducted to develop a ready-to-use (RTU) formulation that would allow storage of the drug product at 2-8° C.

[0351] PEG-FGF-21 (SEQ ID NO:2) was made by site-specifically conjugating a 30 kDa linear PEG moiety containing a distal methoxy group depicted in FIG. 6 to mutant FGF-21 (SEQ ID NO:1), resulting in the molecular conjugate structure shown in FIG. 6, in which para-acetyl-phenylalanine has an L conformation. To achieve site-specific PEGylation, activated PEG was reacted with para-acetyl-L-phenylalanine (pAF), a non-natural amino acid, at position 109 of the FGF-21 molecule to form a chemically stable oxime-linked PEG-protein conjugate by methods known in the art. Activated PEG is commercially available from a number of sources, including, for example, NOF Corporation, Tokyo, Japan.

[0352] Decreasing the pH of the formulation was shown to increase the aggregation rate of the molecule, while increasing the pH of the formulation was shown to increase the deamidation rate. At a target protein concentration of 10 mg / mL, pH 7.0 was found to be the best balance between aggregation and deamidation rates. Increasing the sucrose concentration was shown to further stabilize the molecule against aggregation.

[0353] The formulation selected for further study was PEG-FGF-21 at 10 mg / mL in 20 mM L-histidine, 600 mM sucrose, pH 7.0. With an expected weekly dose of 20 mg per patient, a highly concentrated drug product was developed in a pre-filled syringe. Further optimization of the formulation was required to allow for a highly concentrated drug product.

[0354] Initial development of high concentration formulations was performed in 3 mL type I glass vials. Upon stirring 15 mg / mL and 22 mg / mL PEG-FGF-21 in 20 mM L-histidine, 600 mM sucrose, pH 7.0, air bubble entrapment was observed and the solution became increasingly turbid (Figure 5, left image). In an attempt to alleviate the problem, polysorbate 80 was used. Polysorbate 80 was able to prevent air bubble entrapment (Figure 5, right image). Thus, polysorbate 80 was added to the high concentration formulation of PEG-FGF-21 at a concentration of 0.05% (w / v).

[0355] Oxidation is a critical quality characteristic of PEG-FGF-21, and oxidation of methionine 169 near the C-terminus abolishes receptor binding and therefore activity of the molecule. Metal-catalyzed oxidation is one of the mechanisms by which proteins can be oxidized. Trace concentrations of metals can be present in protein formulations due to carryover from cell culture, contact with stainless steel vessels, impurities in excipients, the process of forming prefillable syringes, etc.

[0356] To examine whether PEG-FGF-21 was susceptible to metal-catalyzed oxidation, samples were incubated with and without the addition of a metal cocktail (250 ppm Fe, 10 ppm Cu, 15 ppm Cr, 15 ppm Ni, 10 ppm Mo) in the presence or absence of 0.05 mM pentetic acid (diethylenetriaminepentaacetic acid, DTPA) for 1 month at 25° C. and for 2 weeks at 40° C., respectively. Oxidation levels of methionine 1 and methionine 169 were determined by tryptic peptide mapping (FIGS. 2A and 2B).

[0357] In the absence of pentetic acid (DTPA), significant oxidation of both methionine residues was observed even in samples without added metals, while the oxidation level was even higher in samples spiked with the metal cocktail. No increase in oxidation level was observed for either methionine in samples with 50 μM pentetic acid. This suggests that PEG-FGF-21 is sensitive to metal-catalyzed oxidation and that trace levels of metals are already present in the formulation. Pentetic acid effectively chelated the metals present in the formulation as well as additional metals spiked into the formulation, preventing metal-catalyzed oxidation of methionines 1 and 169. Thus, 50 μM pentetic acid was added to a high concentration formulation of PEG-FGF-21.

[0358] To optimize the pH for the high concentration formulation, we re-examined the aggregation data for PEG-FGF-21 collected between pH 6.3 and 8.5, which showed that below pH 6.8, the aggregation rate rapidly increased (Figures 3A and 3B). Thus, we increased the target pH for the high concentration formulation from pH 7.0 to pH 7.1.

[0359] To compare the new high concentration formulation with existing formulations, PEG-FGF-21 at 20 mg / mL in 20 mM L-histidine, 600 mM sucrose, 0.05 mM pentetic acid, 0.05% (w / v) polysorbate 80, pH 7.0 (before pH optimization) was subjected to stability studies and compared to PEG-FGF-21 at 22.5 mg / mL in 20 mM L-histidine, 600 mM sucrose, pH 7.0.

[0360] As shown in Figures 4A and 4B and Figures 1A and 1B, the addition of polysorbate 80 and pentetic acid to PEG-FGF-21 formulations resulted in a decrease in aggregation rates at all temperatures and a decrease in deamidation at 40°C. Test Method

[0361] Aggregation: Aggregation was examined by size-exclusion high performance liquid chromatography on a commercial system (e.g., Agilent Technologies 1100 series HPLC system with PDA detector or Waters Alliance e2695 series HPLC with PDA detector) equipped with a commercial analytical column (Tosoh TSK gel G3000SWXL, 7.8×300 mm, P / N: 08541). High molecular weight species were separated from protein monomers using a mobile phase of 95% phosphate buffered saline (PBS) and 5% ethanol at a flow rate of 0.8 ml / min. Samples were diluted with PBS to a protein concentration of 0.5 mg / mL, and 0.01 mg (0.02 mL) was injected for each experiment. The amount of aggregates was determined by dividing the area of ​​the high molecular weight peak by the total area of ​​all peaks observed using the instrument software.

[0362] Deamidation: Deamidation was examined by anion-exchange high performance liquid chromatography on a commercial system (e.g., Agilent Technologies 1100 series HPLC system with PDA detector or Waters Alliance e2695 series HPLC with PDA detector) equipped with a commercial analytical column (Agilent Bio WAX, non-porous, 5 μm column, 4.6×250 mm, P / N: 5190-2487). Mobile phase A consisted of 20 mM Tris, pH 8.2, and mobile phase B consisted of 20 mM Tris, 500 mM sodium chloride, pH 8.2. Charged protein variants were separated using a linear gradient from 2% B to 67% B over 20 min at a flow rate of 1.0 ml / min. Samples were diluted with mobile phase A to a protein concentration of 1 mg / mL, and 0.075 mg (0.075 mL) was injected for each experiment. The amount of deamidation was determined by dividing the area of ​​the acidic variant peak by the total area of ​​all peaks observed using the instrument software.

[0363] Oxidation: Oxidation was investigated by tryptic peptide mapping. Protein samples were digested with trypsin, and the resulting peptides were separated by reversed-phase ultra-performance liquid chromatography on a commercial system (e.g., Waters Acquity UPLC) equipped with a commercial analytical column (Waters Acquity C18 BEH Peptide RP UPLC column, 2.1 x 150 mm, particle size: 1.7 μm, pore size: 130 Å, P / N: 186003556). Mobile phase A consisted of 0.2% trifluoroacetic acid (TFA) in water, and mobile phase B consisted of 0.2% TFA in acetonitrile. Tryptic peptides were separated using a composite gradient from 10% B to 40% B over 27 min at a temperature of 60 °C and a flow rate of 0.3 ml / min. The amount of oxidation was determined by dividing the area of ​​the oxidized peptide peak by the total area of ​​the oxidized peptide peak and the area of ​​the corresponding nonoxidized peptide peak using the instrument's software.

[0364] Particulate formation and bubble formation: Visual observation is performed.

[0365] Concentration of PEG-FGF-21: The concentration of PEG-FGF-21 in the pharmaceutical formulations disclosed herein was measured by Slope Spectroscopy, e.g., Agilent Cary 60 UV-Vis spectrometer equipped with SoloVPE Fibrette (C Technologies, Inc.; P / NOF0002-P50) (SoloVPE disposable UV plastic vessel (C Technologies, Inc.; P / NOC0009-C1)) using an attenuation constant of 0.87 (mL / (mg×cm)) at 280 nm. Throughout this disclosure, the dosage of PEG-FGF-21 is based on the pharmaceutical formulation thus measured. Slope Spectroscopy is also applicable not only to PEG-FGF-21, but generally to any FGF-21 conjugate.

[0366] Determination of pH: pH was determined according to standard methods (USP <791> ) was determined.

[0367] Excipient concentration: The concentration of each excipient (e.g., DTPA, PS80, histidine, sucrose) in the pharmaceutical formulations disclosed herein was determined / calculated to be the amount (weight, moles, etc.) of each excipient added to the pharmaceutical formulation during its manufacture per final volume unit of the finished pharmaceutical formulation. Alternatively, the excipient concentration may be based on the actual amount of each excipient in the pharmaceutical formulation.

Claims

1. (i) FGF-21 conjugate; (ii) histidine at a concentration between about 10 mM and about 50 mM; (iii) sucrose at a concentration between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration between about 10 μM and about 100 μM; A pharmaceutical formulation comprising: the pH of the formulation is between about 6.7 and about 7.5; The FGF-21 conjugate has formula I: 【Chemistry 1】 [In the formula, n is between about 670 and about 690 (e.g., about 681); The FGF-21 polypeptide comprises SEQ ID NO:

1. (eg, an FGF-21 conjugate corresponding to the compound of SEQ ID NO: 2, 4, or 6).

2. the FGF-21 conjugate is present at a concentration of about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, or about 40 mg / ml; (i) histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; (ii) histidine at a concentration of 20 mM; sucrose at a concentration of 600 mM; polysorbate 80 at a concentration of 0.05% (w / v); and DTPA at a concentration of 50 μM, and having a pH of 7.1; (iii) FGF-21 conjugate at a concentration of about 10 mg / mL; histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; (iv) FGF-21 conjugate at a concentration of about 20 mg / mL; histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; or (v) FGF-21 conjugate at a concentration of 10 mg / mL; histidine at a concentration of 20 mM; sucrose at a concentration of 600 mM; polysorbate 80 at a concentration of 0.05% (w / v); and DTPA at a concentration of 50 μM, and having a pH of 7.0; 2. The pharmaceutical formulation of claim 1.

3. 1. A pharmaceutical formulation comprising an FGF-21 conjugate of SEQ ID NO: 2, 4, or 6 at a concentration of about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, or about 40 mg / ml; histidine at a concentration of 20 mM; sucrose at a concentration of 600 mM; polysorbate 80 at a concentration of 0.05% (w / v); and DTPA at a concentration of 50 μM, and having a pH of 7.

0.

4. The pharmaceutical formulation of any one of claims 1 to 3, wherein the formulation is formulated for subcutaneous administration (eg subcutaneous administration using a safety syringe).

5. A pharmaceutical formulation according to any one of claims 1 to 4 for use in the treatment or prevention of a disease or condition associated with fibrosis and / or diabetes in a subject.

6. 6. The pharmaceutical formulation of claim 5, wherein the disease or condition is diabetes.

7. 7. The pharmaceutical formulation of claim 6, wherein the diabetes is type 2 diabetes.

8. 6. The pharmaceutical formulation of claim 5, wherein the disease or condition is non-alcoholic steatohepatitis (NASH).

9. 9. The pharmaceutical formulation of any one of claims 5 to 8, wherein administration of an effective amount of the pharmaceutical formulation to a subject results in reduced liver stiffness, reduced body fat percentage, reduced body weight, reduced liver weight to body weight ratio, reduced liver lipid content, reduced liver fibrosis area, reduced fasting blood glucose levels, reduced fasting triglyceride levels, reduced LDL cholesterol levels, reduced ApoB levels, reduced ApoC levels, increased HDL cholesterol, or any combination thereof.

10. The pharmaceutical formulation of any one of claims 5 to 9, wherein the FGF-21 conjugate is administered in a flat dose of about 20 mg.

11. The pharmaceutical formulation according to any one of claims 5 to 10, wherein the FGF-21 conjugate is administered at weekly dosing intervals.

12. The pharmaceutical formulation according to any one of claims 5 to 11, wherein the pharmaceutical formulation is administered subcutaneously (eg administered subcutaneously using a safety syringe).

13. The pharmaceutical formulation of any one of claims 5 to 12, wherein administration of the pharmaceutical formulation to a subject results in a reduced level of liver fat; a reduced level of liver damage; a reduced level of fibrosis; a reduction in serum Pro-C3 (N-terminal type III collagen propeptide), a biomarker of fibrosis; a reduction in alanine aminotransferase (ALT) levels; a reduction in aspartate aminotransferase (AST) levels; an increase in serum adiponectin levels; a reduction in plasma LDL levels; an increase in plasma HDL levels; a reduction in plasma triglyceride levels; a reduction in liver stiffness levels; or any combination thereof, compared to levels in an untreated subject or in a subject prior to administration of the pharmaceutical formulation.

14. (i) FGF-21 conjugate; (ii) histidine at a concentration between about 10 mM and about 50 mM; (iii) sucrose at a concentration between about 100 mM and about 1 M; (iv) polysorbate 80 at a concentration of between about 0.01% and about 0.1% (w / v); and (v) DTPA at a concentration between about 10 μM and about 100 μM A vial containing a formulation comprising the pH of the formulation is between about 6.7 and about 7.5; The FGF-21 conjugate has formula I: 【Chemistry 2】 [In the formula, n is between about 670 and about 690 (e.g., about 681); The FGF-21 polypeptide comprises SEQ ID NO:

1. (e.g., an FGF-21 conjugate corresponding to the compound of SEQ ID NO: 2, 4, or 6).

15. The formulation comprises: (i) about 10 mg to about 20 mg of FGF-21 conjugate; histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; (ii) about 10 mg of FGF-21 conjugate; histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; or (iii) about 20 mg of FGF-21 conjugate; histidine at a concentration of about 20 mM; sucrose at a concentration of about 600 mM; polysorbate 80 at a concentration of about 0.05% (w / v); and DTPA at a concentration of about 50 μM, and having a pH of about 7.1; 15. The vial of claim 14.

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