FGF21 preparations

Abstract

The present application provides a pharmaceutical formulation comprising an FGF-21 polypeptide, for example, a modified FGF-21 polypeptide, such as PEG-FGF-21, and one or more stabilizers, such as chelator DPTA. The formulation may be further stabilized by including a surfactant, such as polysorbate 80, and / or adjusting the pH to about 7.1. Additionally, a method of preparation, a method of treatment, and a kit are provided.

Description

Technology Field

[0001] Reference to the list of sequences electronically submitted via EFS-WEB

[0002] The contents of the electronically submitted sequence list (filename: 3338_133PC01_SeqListing.txt; size: 5,532 bytes; and creation date: June 26, 2019) submitted together with this application are incorporated herein by reference in their entirety.

[0003] background

[0004] field

[0005] The present application relates, among other things, to a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, which is stabilized using a chelator (e.g., DTPA) and a surfactant (e.g., PS80) and has a specific pH range (e.g., about 7.1), for administration via various routes, including, e.g., subcutaneous administration. Background Technology

[0006] Fibroblast growth factor (FGF) is a polypeptide that is widely expressed in developing and adult tissues and plays a critical role in numerous physiological functions (Baird et al., Cancer Cells, 3:239-243, 1991) (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)).

[0007] FGF-21 can be used to treat diseases and conditions associated with fibrosis. Fibrosis is the formation of an excessive amount of fibrous connective tissue in organs or tissues. The excessive deposition of fibrous tissue is associated with pathological conditions that can lead to impairment of organ or tissue function. Affected organs may include the lungs (pulmonary fibrosis), liver (hepatic fibrosis), kidneys (renal fibrosis), and heart (cardiac fibrosis). Fibrosis can also affect other tissues and organs, including joints, skin, intestines, bone marrow, etc. Exemplary fibrotic conditions or diseases include, but are not limited to, non-alcoholic steatohepatitis (NASH) affecting the liver; diabetic nephropathy and diabetic kidney disease affecting the kidneys; and metabolic heart failure affecting the heart. For example, NASH is characterized by fat accumulation, inflammation, and damage to the liver in people who do not or rarely consume alcohol, and the disease can lead to liver cirrhosis. NASH tends to be diagnosed in overweight or obese middle-aged people with elevated blood levels of certain lipids and diabetes or prediabetes.

[0008] FGF-21 tends to be proteolytic in vivo, form aggregates in vitro, and undergo 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; WO 2006 / 0065582), which has been reported to potentially limit 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)]).

[0009] The present disclosure addresses, above all, the need for a pharmaceutical formulation that solves problems associated with the manufacture of pharmaceutical formulations comprising FGF-21 polypeptide, and in particular, modified FGF-21 polypeptide.

[0010] The present disclosure provides a pharmaceutical formulation comprising a fibroblast growth factor 21 (FGF-21) polypeptide, e.g., a modified FGF-21 polypeptide, and an aminopolycarboxylic acid cation chelator, e.g., diethylenetriaminepentaacetic acid (also known as pentetic acid or DTPA), wherein the formulation has improved stability compared to a reference formulation not containing an aminopolycarboxylic acid cation chelator. These pharmaceutical formulations exhibit one or more improvements over alternative formulations known in the art, e.g., a lower deamidation rate when stored at 40°C for about one month, and / or a lower high molecular weight (HMW) aggregation rate when stored at 40°C for about one month.

[0011] The presence of an aminopolycarboxylic acid cation chelator in the disclosed formulation also reduces FGF-21 polypeptide oxidation, and in particular, methionine oxidation. For example, the presence of DTPA in the formulation can reduce or prevent the oxidation of one or more FGF-21 methionines (or corresponding methionines in SEQ ID NO: 1 and 2) corresponding to amino acid 1 and / or amino acid 169 of SEQ ID NO: 3 at 25°C and / or 40°C.

[0012] In some aspects, the DTPA cation chelator is present in amounts of about 10 μM to about 100 μM DTPA, about 20 μM to about 90 μM DTPA, about 30 μM to about 80 μM DTPA, about 25 μM to about 75 μM DTPA, about 40 μM to about 60 μM DTPA, about 30 μM to about 70 μM DTPA, or about 40 μM to about 70 μM DTPA. In other aspects, the DTPA cation chelator is present in amounts of about 40 μM, about 45 μM, about 50 μM, about 55 μM, or about 60 μM DTPA.

[0013] In some aspects of the pharmaceutical formulations disclosed herein, 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.

[0014] In some aspects, the pH is 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 aspects, 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 aspects, the pharmaceutical formulation is more stable than the reference formulation having a pH of 6.5.

[0015] In some aspects, the pharmaceutical formulation disclosed herein further comprises a surfactant, e.g., a nonionic surfactant. In some aspects, the non-anionic surfactant is a polysorbate, e.g., polyoxyethylene (20) sorbitan monooleate (polysorbate 80). In some aspects, the polysorbate 80 surfactant 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).

[0016] In some aspects, the polysorbate 80 surfactant 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 aspects, the surfactant reduces particulate and / or bubble formation, for example, when stirred on a shaker.

[0017] In some aspects, the pharmaceutical formulation disclosed herein further comprises an amino acid buffer, e.g., histidine. In some aspects, the histidine buffer is present in amounts 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 histidine, or about 40 mM to about 60 mM histidine. In some aspects, histidine buffer is present in amounts 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 aspects, the pharmaceutical formulation disclosed herein further comprises an osmotic regulator, e.g., a sugar. In some aspects, the sugar is sucrose. In some aspects, the sucrose osmotic regulator is present in amounts 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 aspects, the sucrose osmotic regulator is present in amounts 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 of the pharmaceutical formulations disclosed herein, the FGF-21 polypeptide comprises, consists of, or is essentially composed of a modified FGF-21 polypeptide. In some aspects, the modified FGF-21 polypeptide comprises a polypeptide 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 with respect to the amino acid sequence of SEQ ID NO: 3, wherein the polypeptide has FGF-21 activity.

[0020] In some aspects, the modified FGF-21 polypeptide is linked to a half-life extension moiety. In some aspects, the modified FGF-21 polypeptide is linked to a half-life extension moiety through a side chain of non-naturally encoded aminos within the sequence of the FGF-21 polypeptide. In some aspects, the half-life extension moiety comprises albumin, an immunoglobulin constant region or part thereof, an immunoglobulin-binding polypeptide, immunoglobulin G (IgG), an albumin-binding polypeptide (ABP), a PAS-modified moiety, an HES-modified moiety, an XTEN, an Fc region, and any combination thereof. In some aspects, the half-life extension moiety comprises a water-soluble polymer. In some aspects, the water-soluble polymer comprises polyethylene glycol (PEG). In some aspects, the PEG has an average molecular weight of about 10 kDa to about 40 kDa. In some respects, PEG has an average molecular weight of about 30 kDa.

[0021] In some aspects of the formulations disclosed herein, the non-naturally coded amino acid is a phenylalanine derivative. In some aspects, the phenylalanine derivative is para-acetyl-L-phenylalanine. In some aspects, a half-life extension moiety is linked to the non-naturally coded amino acid via an oxime linkage. In some aspects, the non-naturally coded amino acid replaces the amino acid glutamine 109 of SEQ ID NO: 3.

[0022] In some aspects of the formulations disclosed herein, FGF-21 polypeptide is present at a concentration of about 1 mg / ml to about 40 mg / ml. In some aspects, FGF-21 polypeptide is present at a concentration of about 10 mg / ml or about 20 mg / ml. In some aspects, FGF-21 polypeptide is present in an amount of about 1 mg to about 40 mg per dose. In some aspects, FGF-21 polypeptide is present in an amount of about 1 mg, about 5 mg, about 10 mg, about 20 mg, or about 40 mg per dose.

[0023] 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. In some aspects of the formulations disclosed herein, the FGF-21 polypeptide is PEG-FGF-21 (Sequence Identification No.: 2).

[0024] The present disclosure provides a pharmaceutical formulation comprising: (i) FGF-21 polypeptide; (ii) histidine at a concentration of about 10 mM to about 50 mM; (iii) sucrose at a concentration of about 100 mM to about 1 M; (iv) polysorbate 80 at a concentration of about 0.01% to about 0.1% (w / v); and (v) DTPA at a concentration of about 10 μM to about 100 μM; wherein the pH of the formulation is about 6.7 to about 7.5.

[0025] The present disclosure also provides a pharmaceutical formulation comprising: (i) FGF-21 polypeptide; (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 pH is about 7.1. Additionally, a pharmaceutical formulation comprising: (i) FGF-21 polypeptide; (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.

[0026] The present disclosure also provides a pharmaceutical formulation comprising: (i) an FGF-21 polypeptide; (ii) histidine at a concentration of about 20 mM; and (iii) sucrose at a concentration of about 600 mM; wherein the pH is about 7.0. Additionally, a pharmaceutical formulation comprising: (i) an FGF-21 polypeptide; (ii) histidine at a concentration of 20 mM; and (iii) sucrose at a concentration of 600 mM; wherein the pH is 7.0.

[0027] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF-21 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.

[0028] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF-21 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 pH is about 7.1.

[0029] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF-21 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.

[0030] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF21 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.

[0031] The present disclosure also provides a method for improving the stability of a pharmaceutical formulation comprising an FGF-21 polypeptide, comprising mixing an aminopolycarboxylic acid cation chelator, wherein the formulation has improved stability compared to a reference formulation not containing the aminopolycarboxylic acid cation chelator. In one aspect, the improvement in stability comprises (i) an increase in physical stability, (ii) an increase in chemical stability, or (iii) a combination thereof. In one aspect, the increase in physical stability comprises (i) prevention or reduction of polypeptide aggregation, (ii) prevention or reduction of polypeptide fragmentation, or (iii) a combination thereof. In some aspects, the increase in chemical stability comprises (i) prevention or reduction of deamidation, (ii) prevention or reduction of oxidation, or (iii) a combination thereof. In some aspects, the improvement in stability comprises one or more of the following:

[0032] (a) For the reference formulation, a lower deamidation rate when stored at 40°C for about 1 month;

[0033] (b) for the reference formulation, a lower high molecular weight (HMW) aggregation rate when stored at 40°C for about 1 month; or

[0034] (c) Both (a) and (b).

[0035] In some aspects of the method for improving the stability of the pharmaceutical formulation disclosed herein, an aminopolycarboxylic acid cation chelator reduces the oxidation of one or more methionines corresponding to amino acid 1 and / or amino acid 169 of SEQ ID NO: 3 (or corresponding methionines in SEQ ID NO: 1 or SEQ ID NO: 2, or other modified FGF-21 polypeptides) at 25°C and / or 40°C. In some aspects, the aminopolycarboxylic acid cation chelator is DTPA. In some aspects, the DTPA cation chelator is present in amounts of about 10 μM to about 100 μM DTPA, about 20 μM to about 90 μM DTPA, about 25 μM to about 75 μM DTPA, about 40 μM to about 60 μM DTPA, about 30 μM to about 70 μM DTPA, about 30 μM to about 80 μM DTPA, or about 40 μM to about 70 μM DTPA. In some aspects, the DTPA cation chelator is present in amounts of about 40 μM, about 45 μM, about 50 μM, about 55 μM, or about 60 μM DTPA.

[0036] In some aspects, a method for improving the stability of a pharmaceutical formulation disclosed herein further comprises adjusting the pH of the formulation 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 aspects, the pH is adjusted to a pH value of about 6.8 to about 7.5, or about 6.9 to about 7.4, or about 7.0 to about 7.3, or about 7.1 to 7.2, or about 7.1 to about 7.3, or about 7.1 to about 7.4, or about 7.1 to about 7.5. In some aspects, 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 respects, pH-adjusted formulations are more stable than reference formulations with a pH of 6.5 (i.e., formulations that have the same ingredients at the same concentration but with a different pH).

[0037] In some aspects, a method for improving the stability of the pharmaceutical formulation disclosed herein further comprises mixing a surfactant. In some aspects, the surfactant is a nonionic surfactant, e.g., polysorbate. In some aspects, the polysorbate is polysorbate 80. In some aspects, the polysorbate 80 surfactant 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 aspects, the polysorbate 80 surfactant 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 aspects, the surfactant reduces particulate and / or bubble formation, for example, when stirring the formulation on a shaker.

[0038] In some aspects, a method for improving the stability of the pharmaceutical formulation disclosed herein further comprises mixing an amino acid buffer, e.g., histidine. In some aspects, the histidine buffer is mixed in amounts 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 aspects, histidine buffer is mixed in amounts 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.

[0039] In some aspects, a method for improving the stability of the pharmaceutical formulation disclosed herein further comprises mixing an osmotic regulator, e.g., a sugar. In some aspects, the sugar is sucrose. In some aspects, the sucrose osmotic regulator 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 aspects, sucrose osmotic regulators are mixed in amounts 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.

[0040] In some aspects of the method for improving the stability of the pharmaceutical formulation disclosed herein, the FGF-21 polypeptide is a modified FGF-21 polypeptide. In some aspects, the modified FGF-21 polypeptide comprises an FGF-21 polypeptide 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 with respect to the amino acid sequence of SEQ ID NO: 3, wherein the polypeptide has FGF-21 activity. In some aspects, the modified FGF-21 polypeptide is linked to a half-life extension moiety. In some aspects, the modified FGF-21 polypeptide is linked to a half-life extension moiety through a side chain of non-naturally encoded aminos within the sequence of the FGF-21 polypeptide.

[0041] In some aspects, the half-life extension moiety comprises, for example, albumin, an immunoglobulin constant region or part thereof, an immunoglobulin-binding polypeptide, IgG, ABP, a PAS-modified moiety, a HES-modified moiety, XTEN, an Fc region, and any combination thereof. In some aspects, the half-life extension moiety comprises a water-soluble polymer. In some aspects, the water-soluble polymer is PEG. In some aspects, PEG has an average molecular weight of about 10 kDa to about 40 kDa. In some aspects, PEG has an average molecular weight of about 30 kDa. In some aspects, the non-naturally encoded amino acid is a phenylalanine derivative. In some aspects, the phenylalanine derivative is para-acetyl-L-phenylalanine. In some aspects, the half-life extension moiety is linked to the non-naturally encoded amino acid via an oxime linkage. In some aspects, a non-naturally coded amino acid replaces the amino acid glutamine 109 of sequence identification number: 3. In some aspects, the FGF-21 polypeptide is present at a concentration of about 1 mg / ml to about 40 mg / ml. In some aspects, the FGF-21 polypeptide is present at a concentration of about 10 mg / ml or about 20 mg / ml. In some aspects, the formulation is an aqueous formulation. In some aspects of the method disclosed above, the FGF-21 polypeptide is PEG-FGF-21 (sequence identification number: 2).

[0042] The present disclosure also provides a method for improving the stability of a formulation containing an FGF-21 polypeptide, comprising mixing (i) histidine at a concentration of about 10 mM to about 50 mM; (ii) sucrose at a concentration of about 100 mM to about 1 M; (iii) polysorbate 80 at a concentration of about 0.01% to about 0.1% (w / v); and (iv) DTPA at a concentration of about 10 μM to about 100 μM; wherein the pH of the formulation is about 6.7 to about 7.5. Additionally, a method for improving the stability of a formulation containing an FGF-21 polypeptide comprises mixing (i) histidine at a concentration of about 20 mM; (ii) sucrose at a concentration of about 600 mM; and (iii) polysorbate 80 at a concentration of about 0.05% (w / v). A method is provided comprising (iv) mixing DTPA at a concentration of about 50 μM, wherein the pH is about 7.1. Additionally, a method for improving the stability of a formulation containing FGF-21 polypeptide is provided, 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.

[0043] The present disclosure also provides a method for improving the stability of a formulation containing FGF-21 polypeptide, 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. The present disclosure also provides a method for improving the stability of a formulation containing FGF-21 polypeptide, 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. Additionally, a method for improving the stability of a formulation containing PEG-FGF-21 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 µM; Here, a method is provided in which the pH is approximately 7.1.

[0044] The present disclosure also provides a method for improving the stability of a formulation containing PEG-FGF-21 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 µM; wherein the pH is about 7.1. Additionally, a method for improving the stability of a formulation containing PEG-FGF-21 at a concentration of about 10 mg / mL is provided, 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.

[0045] Additionally, a method for improving the stability of a formulation containing PEG-FGF-21 at a concentration of about 20 mg / mL is provided, 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.

[0046] The present disclosure also provides a pharmaceutical formulation prepared according to any of the methods for improving the stability of a formulation comprising FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21). Additionally, a kit is provided comprising (i) a pharmaceutical formulation comprising FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21), and (ii) instructions for use.

[0047] The present disclosure also provides a method for treating or preventing a disease or condition associated with fibrosis and / or diabetes, comprising administering an effective amount of a pharmaceutical formulation containing FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21) to a subject who requires treatment or prevention of a disease or condition associated with fibrosis and / or diabetes. In some aspects, the disease or condition is diabetes. In some aspects, the diabetes is type 2 diabetes. In some aspects, the disease or condition is non-alcoholic fatty liver disease (NASH). In some aspects, administration of a pharmaceutical formulation containing an effective amount of FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21) to a subject reduces liver stiffness, reduces body fat percentage, reduces body weight, reduces liver-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. In some aspects, about 1 mg to about 40 mg of FGF-21 polypeptide is administered per dose. In some aspects, about 1 mg, about 5 mg, about 10 mg, about 20 mg, or about 40 mg of FGF-21 polypeptide is administered per dose. In some aspects, the pharmaceutical formulation is administered subcutaneously. In some aspects, the pharmaceutical formulation is administered subcutaneously using a safety syringe. In some aspects, pharmaceutical preparations are administered daily or weekly.

[0048] In some aspects, administration of a pharmaceutical formulation comprising FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21) according to the treatment method disclosed herein to a subject results in, compared to levels in an untreated subject or to a subject prior to administration of the pharmaceutical formulation, (i) a decrease in the level of hepatic steatosis; (ii) a decrease in the level of hepatic impairment; (iii) a decrease in the level of fibrosis; (iv) a decrease in the level of the fibrosis biomarker serum Pro-C3 (N-terminal type III collagen propeptide); (v) a decrease in the level of alanine aminotransferase (ALT); (vi) a decrease in the level of aspartate aminotransferase (AST); (vii) an increase in the level of serum adiponectin; (viii) a decrease in the level of plasma LDL, an increase in the level of plasma HDL; (ix) a decrease in the level of plasma triglycerides; (x) a decrease in the level of hepatic stiffness; or (xi) provides any combination of these.

[0049] In some aspects, the pharmaceutical formulation is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of about 10 mg / mL; (ii) an amount of histidine to achieve a final concentration of about 20 mM; (iii) an amount of sucrose to achieve a final concentration of about 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of about 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of about 50 uM; and adjusting the pH to about 7.1.

[0050] In some aspects, the pharmaceutical formulation is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of about 20 mg / mL; (ii) an amount of histidine to achieve a final concentration of about 20 mM; (iii) an amount of sucrose to achieve a final concentration of about 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of about 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of about 50 uM; and adjusting the pH to about 7.1.

[0051] In some aspects, the pharmaceutical formulation is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of 10 mg / mL; (ii) an amount of histidine to achieve a final concentration of 20 mM; (iii) an amount of sucrose to achieve a final concentration of 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of 50 uM; and adjusting the pH to 7.1.

[0052] In some aspects, the pharmaceutical formulation is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of 20 mg / mL; (ii) an amount of histidine to achieve a final concentration of 20 mM; (iii) an amount of sucrose to achieve a final concentration of 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of 50 uM; and adjusting the pH to 7.1. Brief explanation of the drawing

[0053] Figures 1a and 1b represents the deamidation of PEG-FGF-21 as a function of formulation composition. Fig. 1a represents deamidation in formulations without pentetetic acid (DTPA) or polysorbate 80. Fig. 1brepresents deamidation in a formulation containing pentetic acid (DTPA) and polysorbate 80. Figures 2a and 2b This shows the metal-catalyzed oxidation of PEG-FGF-21 polypeptide in methionine 1 (Fig. 2a) and methionine 169 (Fig. 2b) in the presence and absence of 50 μM DTPA (also known as pentetic acid). Figures 3a and 3b This indicates the effect of pH on PEG-FGF-21 aggregation. Fig. 3a It represents aggregation as a function of time, pH, and the buffer system used. Fig. 3b It shows coagulation at different temperatures and times (5℃, 14 months; 25℃, 1 month; 40℃, 1 day) and different pH. Figures 4a and 4b represents the aggregation of PEG-FGF-21 as a function of formulation composition. Fig. 4a It indicates aggregation in formulations without pentetic acid (DTPA) or polysorbate 80. Fig. 4b represents aggregation in a formulation containing pentetic acid (DTPA) and polysorbate 80. Fig. 5 This shows that higher concentrations of PEG-FGF-21 without PS80 became turbid after 24 hours (left) or 6 hours of wrist-action shaking in a 300 rpm orbital shaker (right). The addition of 0.01% to 0.1% (w / v) of polysorbate 80 reduced the turbidity of the sample. Specific details for implementing the invention

[0054] The present disclosure provides a stabilized pharmaceutical formulation comprising a fibroblast growth factor 21 (FGF-21) polypeptide, for example, a modified FGF-21 polypeptide, e.g., PEG-FGF-21 (SEQ ID: 2). It has been observed that the presence of an aminopolycarboxylic acid cation chelator, e.g., DTPA, reduces the oxidation of one or more amino acid residues, e.g., methionine, in the FGF-21 polypeptide. The incorporation of DTPA into the FGF-21 formulation also reduces the rate of deamidation of the FGF-21 polypeptide during storage and also reduces the rate of high molecular weight aggregation during storage. Further stabilization can be achieved by adjusting the pH of the formulation to 7.1. Additionally, the formulation may be further stabilized by the addition of a surfactant, e.g., polysorbate 80.

[0055] The present disclosure also provides a method for manufacturing the disclosed formulation, as well as a formulation manufactured by applying the disclosed method. Additionally, a method for treating or preventing fibrosis-associated diseases, e.g., NASH and diabetes, is provided, comprising administering the disclosed stabilized formulation to a subject requiring treatment or prevention of fibrosis-associated diseases, e.g., NASH and diabetes.

[0056] definition

[0057] To facilitate a better understanding of the present disclosure, specific terms are defined first. As used in this application, each of the following terms will have the meaning described below, except where otherwise explicitly provided herein. Additional definitions are provided throughout this application.

[0058] The present disclosure includes aspects in which exactly one member of the group is present, used, or otherwise related thereto in a given product or process. The present disclosure includes aspects in which one or more or all members of the group are present, used, or otherwise related thereto in a given product or process.

[0059] The singular form includes plural referents unless the context clearly indicates otherwise. The singular form, as well as the terms “one or more” and “at least one,” may be used interchangeably herein. In a specific aspect, the singular form means “single.” In another aspect, the singular form includes “two or more” or “multiple.” Thus, for example, a reference to “FGF-21” or “FGF-21 polypeptide” is a reference to one or more of these proteins, including equivalents known to those skilled in the art, etc.

[0060] Additionally, when used herein, "and / or" should be considered as a specific disclosure of each of two specific features or components having or not having others. Accordingly, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, as used herein in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following aspects: 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 (alone); B (alone); and C (alone).

[0061] As used herein, the term “about” refers to a value or composition that is within an acceptable range of error for a specific value or composition as determined by a person skilled in the art, depending in part on the manner in which the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” may mean within one or more standard deviations according to the practice of the art. Alternatively, “about” may mean a range of up to 20%. Furthermore, particularly in relation to biological systems or processes, the term may mean up to 10 times the size or up to 5 times the value. Where a specific value or composition is provided in this application and claims, unless otherwise noted, the meaning of “about” is to be assumed to be within an acceptable range of error for that specific value or composition. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries beyond and below the stated value. Thus, “about 10-20” means “about 10 to about 20”. Generally, the term "about" can modify the figure to be greater than or less than the mentioned value by, for example, a fluctuation of 10 percent above or below (higher or lower).

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure relates. For example, the literature [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] provides a general dictionary of many terms used in this disclosure to a person skilled in the art.

[0063] It is understood that in parts of this document where an aspect is described using the term "comprising," similar aspects in other ways described using the terms "constituted" and / or "essentially constituted" are also provided.

[0064] Units, prefixes, and symbols are indicated in their accepted forms within the International System of Units (SI). Numerical ranges are inclusive for the number defining the range. Unless otherwise indicated, amino acid sequences are described from left to right in the amino-to-carboxyl orientation. The headings provided herein are not to limit the various aspects of the disclosure, which may be provided by reference to the specification in its entirety. Accordingly, terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0065] Amino acids are referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are described from left to right with the amino-to-carboxyl orientation.

[0066] aggregation : The term “aggregation” refers to the tendency of the polypeptides disclosed herein, e.g., FGF-21 polypeptide, to form high molecular weight complexes by forming non-covalent linkage complexes with other molecules (e.g., other molecules of the same polypeptide). An exemplary method for measuring the formation of aggregates includes analytical size exclusion chromatography as described in the embodiments herein. For example, to identify polypeptides having reduced aggregation, the relative amount of aggregation with respect to a reference compound may be determined. Additionally, for example, to identify formulations in which FGF-21 polypeptide has reduced aggregation, the relative amount of aggregation with respect to a reference formulation may be determined.

[0067] amino acid substitutionThe term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent sequence or reference sequence (e.g., wild-type sequence) with another amino acid residue. Amino acids may be substituted in the parent sequence or reference sequence (e.g., wild-type polypeptide sequence) by, for example, through chemical peptide synthesis or through recombination methods known in the art. Thus, the reference to "substitution at position X" refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, the substitution pattern may be described according to the label AnY, where A is a single-character code corresponding to the amino acid naturally or originally present at position n, and Y is the substituted amino acid residue. In other aspects, the substitution pattern may be described according to the label An(YZ), where A is a single-character code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position n, and Y and Z are alternative substituted amino acid residues that can replace A.

[0068] In the context of the present disclosure, substitutions (even when referred to as amino acid substitutions) are performed at the nucleic acid level, that is, the substitution of an amino acid residue with an alternative amino acid residue is performed by substituting a codon coding for a first amino acid with a codon coding for a second amino acid.

[0069] approximately As used herein, the term “approximately” applied to one or more values ​​of interest refers to a value similar to the reference value mentioned. In a particular aspect, the term “approximately” refers to a range of values ​​(greater than or less than these) within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in both directions of the reference value mentioned, unless otherwise stated or otherwise evident from the context (except where such numbers exceed 100% of possible values).

[0070] related to As used herein in relation to diseases, the term "associated with" means that the symptoms, measurements, characteristics, or condition of the issue are related to the diagnosis, development, presence, or progression of the disease. The association may be causally related to the disease, but is not required to be.

[0071] biological activity : The term “biological activity” applied to the molecules disclosed herein, e.g., FGF-21 polypeptide, means any substance capable of influencing any physical or biochemical properties of any biological system, pathway, molecule, or interaction associated with a living organism. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended for the diagnosis, healing, alleviation, treatment, or prevention of a disease or condition, e.g., a disease or condition associated with fibrosis, in humans or other animals, or for improving the physical or mental well-being of humans or animals in any other way.

[0072] Conservative amino acid substitution : "Conservative amino acid substitution" is the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), non-charged 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), have been defined in the art. Therefore, when an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, the amino acid substitution is considered conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in the sequence and / or composition of the side chain family members.

[0073] Non-conservative amino acid substitutions include (i) a residue having a positive side chain (e.g., Arg, His, or Lys) being substituted or replaced by a negative residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) being substituted or replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) a cysteine ​​or proline being substituted or replaced by any other residue; or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) being substituted or replaced by a residue having a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly).

[0074] Other amino acid substitutions may also be used. For example, for the amino acid alanine, the substitution may be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine, and D-cysteine. For lysine, it may be replaced with any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D-ornithine. Generally, substitutions in functionally important regions that are expected to induce changes in the properties of the isolated polypeptide include (i) a polar residue, e.g., serine or threonine, being substituted with (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine ​​residue being substituted with (or by) any other residue; (iii) residues having a positive side chain, e.g., lysine, arginine, or histidine, substituted with (or by) a residue having a negative side chain, e.g., glutamic acid or aspartic acid; or (iv) residues having a bulky side chain, e.g., phenylalanine, substituted with (or by) a residue not having such a side chain, e.g., glycine. The possibility that one of the above non-conservative substitutions may alter the functional properties of the protein also correlates with the location of the substitution in functionally important regions of the protein (thus, some non-conservative substitutions may have little to no effect on biological properties).

[0075] preserved As used herein, the term “conserved” refers to amino acid residues of a polypeptide sequence that appear unchanged at the same position in each of two or more sequences being compared. Relatively conserved amino acids are those that are conserved between more related sequences than nucleotides or amino acids appearing elsewhere in the sequence.

[0076] In some aspects, two or more sequences are referred to as "completely conserved" or "identical" when they are 100% identical to each other. In some aspects, two or more sequences are referred to as "highly conserved" when they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some aspects, two or more sequences are referred to as "highly conserved" when they are approximately 70% identical, approximately 80% identical, approximately 90% identical, approximately 95%, approximately 98%, or approximately 99% identical to each other. In some aspects, two or more sequences are referred to as "conserved" when 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 respects, two or more sequences are referred to as "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. Conservation of sequences may apply to the entire length of a polynucleotide or polypeptide, or to a part, region, or feature thereof.

[0077] DeamidationThe term "deamidation" refers to the tendency of amino acid residues within a polypeptide to undergo a deamidation reaction, thereby altering the chemical structure of the amino acids and potentially affecting the function of the polypeptide. Exemplary methods for measuring deamidation are disclosed in the embodiments of this invention. For example, to identify polypeptides having reduced deamidation, the relative amount of deamidation with respect to a reference compound may be determined. Additionally, for example, to identify formulations of FGF-21 polypeptide having reduced deamidation, the relative amount of deamidation with respect to a reference formulation may be determined.

[0078] Diseases associated with fibrosis The term "diseases associated with fibrosis" includes diseases, disorders, and pathological conditions in which fibrosis is observed to be present, or in which fibrosis is known or considered to be associated with the etiology, progression, or symptoms of the disease, or in which fibrosis is known or considered to appear as a result of the progression of the disease.

[0079] Fibrosis can affect organs and tissues such as the pancreas, lungs, heart, kidneys, liver, eyes, nervous system, bone marrow, lymph nodes, endocardial myocardium, or retroperitoneal space. Exemplary diseases associated with fibrosis include non-alcoholic steatohepatitis (NASH), hepatic fibrosis, pre-cirrhosis, cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, pulmonary fibrosis, progressive macrofibrosis, idiopathic pulmonary fibrosis, infusion fibrosis, renal fibrosis, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myelofibrosis, heart failure, metabolic heart failure, cardiac fibrosis, cataract fibrosis, cataract, ocular scarring, pancreatic fibrosis, cutaneous fibrosis, intestinal fibrosis, intestinal stenosis, endocardial cardiomyopathy, atrial fibrosis, mediastinal fibrosis, Crohn's disease, retroperitoneal fibrosis, keloid, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, arthral fibrosis, Peyronie's syndrome, Dupeytren's contracture, diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, steatohepatitis, viral hepatitis, biliary tract disease. Primary hemochromatosis, drug-related cirrhosis, latent cirrhosis, Wilson's disease, and, alpha 1-antitrypsin deficiency, interstitial lung disease (ILD), human fibrotic lung disease, macular degeneration, retinoretinopathy, vitreoretinopathy, myocardial fibrosis, Graves' eye disease, drug-induced ergot poisoning, cardiovascular disease, atherosclerosis / recurrent stenosis, hypertrophic scarring, primary or idiopathic myelofibrosis, and inflammatory bowel disease (including but not limited to collagenous colitis). In some aspects, diseases associated with fibrosis may include hepatic fibrosis, renal fibrosis, pulmonary fibrosis, and cardiac fibrosis. In some aspects, diseases associated with fibrosis may be hepatic fibrosis. In some aspects, diseases associated with fibrosis may be NASH.

[0080] Effective amount : As used herein, the term “effective dose” of the FGF-21 formulation disclosed herein refers to an amount sufficient to achieve a beneficial or desired result, e.g., a clinical result, and as such, the “effective dose” varies depending on the context in which it is applied. For example, in the context of the administration of FGF-21 polypeptide for the treatment of NASH, the effective dose of FGF-21 polypeptide is an amount sufficient to improve, e.g., hepatic fat, liver damage, or fibrosis (e.g., a reduction in hepatic fat, liver damage, or fibrosis relative to levels in untreated subjects or levels prior to the administration of treatment).

[0081] In some aspects, an effective amount of FGF-21 polypeptide for treating NASH, e.g., a modified FGF-21 polypeptide, may alter the levels of one or more fibrosis biomarkers as follows, with respect to levels in untreated subjects or levels in subjects prior to administration of treatment: e.g., decrease in serum Pro-C3; decrease in ALT or AST; increase in serum adiponectin; decrease in plasma LDL; increase in plasma HDL; decrease in plasma triglyceride levels, or any combination thereof.

[0082] The term "effective amount" may be used interchangeably with "effective dose," "therapeutic effective amount," or "therapeutic effective dose."

[0083] FGF-21 activity The term "FGF-21 activity" refers to at least one biological activity of the FGF-21 polypeptide. The term "biological activity" refers to the ability of a molecule, e.g., the FGF-21 polypeptide, to influence any physical or biochemical properties of a biological system, pathway, molecule, or interaction associated with an organism, including but not limited to viruses, bacteria, bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans.

[0084] For example, in the context of unmodified or modified FGF-21, biological activity includes any of the biological functions performed by FGF-21. An exemplary method for determining whether a molecule has at least one biological activity of wild-type FGF-21 (e.g., the wild-type FGF-21 polypeptide of SEQ ID No. 3) may include any functional test known in the art, including the method disclosed in Examples 5 and 17 of U.S. Application Publication No. 2017 / 0189486, the full text of which is incorporated herein by reference.

[0085] For example, the EC of a reference compound having biological activity or having sufficiently high biological activity for intended therapeutic use. 50 Less than 5 times, less than 10 times, less than 20 times, less than 50 times, or less than 100 times higher EC 50 To identify polypeptides having [ ], the relative level of biological activity with respect to a reference compound may be determined. Additionally, for example, the EC of the FGF-21 polypeptide in the reference formulation, for example, that the FGF-21 polypeptide has biological activity or sufficiently high biological activity for intended therapeutic use, may be determined. 50 Less than 5 times, less than 10 times, less than 20 times, less than 50 times, or less than 100 times higher EC 50 To identify preparations having [the substance], the relative level of biological activity with respect to a reference preparation can be determined.

[0086] The reference compounds described herein may be modified, for example, FGF-21 sequences without the modifications described herein. For example, the reference compounds may be wild-type FGF-21 or identical modified FGF-21 polypeptide sequences without a fusion partner, for example, without PEG. Exemplary reference compounds for PEG-FGF-21 include, but are not limited to, the wild-type FGF-21 polypeptide of SEQ ID NO: 3 and the modified FGF-21 polypeptide of SEQ ID NO: 1.

[0087] In some aspects, the reference compound may contain at least one non-naturally coded amino acid that can be linked to a linker, polymer, biologically active molecule, peptide, polypeptide, or half-life extension moiety (e.g., PEG) described herein. In some aspects, the reference compound may contain at least one non-naturally coded amino acid that cannot be linked to a linker, polymer, biologically active molecule, peptide, polypeptide, or half-life extension moiety (e.g., PEG) described herein. In some aspects, the reference compound may contain additional amino acid substitutions, deletions, and / or insertions. In some aspects, a comparison may be performed with a PEGylated or non-PEGylated form of the polypeptide; in the former case, a comparison may be performed with a polypeptide that contains or does not contain the non-naturally coded amino acid.

[0088] FGF-21 polypeptide The term "FGF-21 polypeptide" refers to wild-type FGF-21 polypeptide and modified FGF-21 polypeptide.

[0089] Half-life extension motivator: The term “half-life extension moiety” refers to a pharmaceutically acceptable moiety, domain, or molecule that is covalently linked (“conjugated” or “fused”) to the FGF-21 polypeptide, e.g., the modified FGF-21 polypeptide described herein, either directly or via a linker, through optionally non-naturally encoded amino acids, to prevent or mitigate in vivo proteolytic degradation or other activity-reducing chemical modifications of the modified FGF-21 polypeptide compared to reference compounds such as the unconjugated form of the modified FGF-21 polypeptide or the wild-type FGF-21 polypeptide, increases the half-life, and / or improves or alters other 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 the modified FGF-21 polypeptide, increased manufacturability, and / or reduced immunogenicity of the modified FGF-21 polypeptide.

[0090] sameness As used herein, the term “identity” refers to the preservation of the entire monomer between polymer molecules, for example, between polypeptide molecules. The term “identical” without any additional modifiers (e.g., Protein A is identical to Protein B) means that the sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, “70% identical” is equivalent to describing them as having, for example, “70% sequence identity.”

[0091] The calculation of the percentage identity of two polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison (for example, a gap may be introduced in one or both of the first and polypeptide sequences for optimal alignment, and non-identical sequences may be ignored for comparison). In a specific aspect, the length of the sequence 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. Subsequently, amino acids at corresponding amino acid positions are compared.

[0092] 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 that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the length and number of gaps that must be introduced for the optimal alignment of the two sequences. The comparison of sequences and the determination of the percentage identity between two sequences can be achieved using mathematical algorithms.

[0093] Suitable software programs are available from various sources for the alignment of both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). bl2seq performs a comparison between two sequences using the BLASTN or BLASTP algorithms. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) (www.ebi.ac.uk / Tools / psa). Sequence alignment can be performed using methods known in the relevant technical field, such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

[0094] Different regions within a single polypeptide target sequence aligned with a polypeptide reference sequence may each have their own percent sequence identity. It is noted that percent sequence identity values ​​are rounded to the first decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are rounded to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded to 80.2. Additionally, it is noted that length values ​​are always integers.

[0095] In a specific aspect, the percentage identity (%ID) of the first amino acid sequence with respect to the second amino acid sequence is calculated as %ID = 100 x (Y / Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (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 length of the second sequence, the percentage identity of the first sequence with respect to the second sequence will be higher than the percentage identity of the second sequence with respect to the first sequence.

[0096] A person skilled in the art will recognize that the generation of sequence alignments for calculating percent sequence identity is not limited to binary sequence-sequence comparisons driven solely by primary sequence data. Furthermore, they will recognize that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is T-Coffee, available at www.tcoffee.org, which is also available, for example, from EBI. They will recognize that the final alignment used to calculate percent sequence identity can be curated automatically or manually.

[0097] In vivo proteolytic degradationThe term "in vivo proteolytic degradation" refers to the cleavage of a polypeptide that may result from proteases occurring in an organism upon introduction into a living system (e.g., upon injection into the organism). Proteolysis can potentially affect the biological activity or half-life of the polypeptide. For example, wild-type FGF-21 may be subjected to cleavage at the C-terminus, which produces a terminally cleaved, inactive polypeptide. An exemplary method for measuring the in vivo proteolysis of FGF-21 is the Meso Scale Discovery (MSD)-based electrochemiluminescence immunosorbent assay (ECLIA) described in Example 10 of U.S. Application Publication No. US2017 / 0189486. For example, to identify polypeptides with reduced in vivo proteolysis, the relative amount of in vivo or in vitro proteolysis with respect to a reference compound may be determined. In addition, that is, to identify preparations having reduced in vitro proteolysis of FGF-21 polypeptide, the relative amount of in vivo proteolysis with respect to a reference preparation can be determined.

[0098] isolated As used herein, the term “isolated” refers to a substance or entity (e.g., polypeptide) separated from at least a portion of the components that were associated (naturally or in an experimental setting). The isolated substances (e.g., proteins) may have varying levels of purity relative to the substances to which they were associated.

[0099] connectedAs used herein, the terms “linked” or “attached” refer to an amino acid sequence covalently or non-covalently joined to a molecule, e.g., a water-soluble polymer. The term “linked” means the conjugation or chaining of an amino acid sequence at the C-terminus or N-terminus (e.g., FGF-21 polypeptide) and a second amino acid sequence (e.g., albumin half-life extender). This also includes the insertion of any two amino acids into the second amino acid sequence (e.g., FGF-21 polypeptide) of the entire amino acid sequence (e.g., XTEN half-life extender). In some aspects, linkage may occur between a side chain of an amino acid within the first amino acid sequence (e.g., FGF-21 polypeptide) and a second molecule (e.g., the second amino acid sequence or a soluble polymer, e.g., PEG). In the context of this disclosure, the terms “fused” or “fused” indicate that at least two polypeptide chains are operably linked and recombinantly expressed. In some aspects, two polypeptide chains may be "fused" as a result of chemical synthesis. In the context of this disclosure, the terms "conjugate" or "conjugation" indicate that two molecular entities (e.g., two polypeptides, or a polypeptide and a polymer, e.g., PEG) are chemically linked.

[0100] Modified FGF-21 polypeptideAs used herein, "modified FGF-21 polypeptide," "modified fibroblast growth factor 21," or "modified FGF-21" and their unhyphenated forms are used interchangeably and will include polypeptides and proteins that differ from wild-type FGF-21 (e.g., wild-type humFGF-21 of SEQ ID No. 3) and typically have at least one biological activity of fibroblast growth factor 21, as well as, regardless of their biological activity, FGF-21 analogs, FGF-21 isoforms, FGF-21 mimics, FGF-21 fragments, hybrid FGF-21 proteins, fusion proteins, oligomers and multimers, homologues, glycosylated pattern modifieds, modifieds, splice modifieds, and their mutant proteins. Modified FGF-21 polypeptides included in this definition are described in more detail below.

[0101] Mutation : In the content of this disclosure, the terms “mutation” and “amino acid substitution” (sometimes referred to simply as “substitution”) as defined above are considered interchangeable.

[0102] unnaturally encoded amino acids"Non-naturally coded amino acid" refers to an amino acid that is not one of the 20 common amino acids, pyrrolicine, or selenocysteine. Other terms that may be used synonyms with the term "non-naturally coded amino acid" include "non-natural amino acid," "non-natural amino acid," "non-naturally occurring amino acid," and various hyphenated and hyphenated versions thereof. The term "non-naturally coded amino acid" also includes, but is not limited to, amino acids that are represented by a modification (e.g., post-translational modification) of a naturally coded amino acid (including, but not limited to, the 20 common amino acids), but which are not naturally incorporated into the polypeptide chain that grows by the translation complex. Examples of such non-naturally coded amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In one specific aspect of the present disclosure, the non-naturally encoded amino acid is para-acetyl-L-phenylalanine.

[0103] patient As used herein, "patient" refers to a subject who is seeking or may need treatment, is requesting treatment, is accepting treatment, or will accept treatment, or is being managed by a specialist trained in a specific disease or condition.

[0104] Pharmaceutical composition : The term “pharmaceutical composition” refers to a preparation in which the biological activity of the active ingredient (FGF-21 polypeptide, e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) is made effective, and which does not contain any additional ingredients (e.g., excipients and water) that are unacceptably toxic to the subject to whom the composition is administered. Such composition may be sterile.

[0105] Constraints allowableThe term "pharmaceutical acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the scope of sound medical judgment and in accordance with a reasonable benefit / risk ratio. Generally, approval by a federal or state regulatory agency for use in animals, and more particularly in humans (or listed in the United States Pharmacopoeia or other commonly recognized pharmacopoeias), means that these compounds, substances, compositions, and / or dosage forms are pharmaceutically acceptable. Compounds, substances, compositions, and / or dosage forms that are generally acceptable as safe for therapeutic purposes are "therapeutically acceptable."

[0106] Pharmaceutically acceptable excipients As used herein, the phrase “pharmaceuticalally acceptable excipients” refers to any component other than the active compound described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is substantially non-toxic and non-inflammatory in the subject. Excipients may include, for example, chelators, surfactants, buffers, osmoregulators, antioxidants, emulsifiers, fillers (diluents), preservatives, adsorbents, suspending or dispersing agents, sweeteners, and hydrated water. Excipients generally accepted as safe for therapeutic purposes are “therapeutically acceptable excipients.”

[0107] Pharmaceutically acceptable salt: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which 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 pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids.

[0108] polypeptide : The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may contain modified amino acids. The term also includes amino acid polymers modified naturally or by intervention; e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, e.g., conjugation with a labeling component. Additionally, polypeptides containing one or more analogs of amino acids (e.g., non-natural amino acids, e.g., homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art, are included within the definition.

[0109] As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologs, paralogs, fragments thereof, and other equivalents, modifications, and analogs. Polypeptides may be single polypeptides or multimolecular complexes, such as dimers, trimers, or tetramers. They may also include single-stranded or multi-stranded polypeptides. Most commonly, disulfide linkages appear in multi-stranded polypeptides. The term polypeptide may also be applied 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, “peptide” may have a length of 50 or fewer amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0110] prevention As used herein, the term “prevention” 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, features, or clinical manifestations of a specific disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or manifestations of a specific disease, disorder, and / or condition; partially or completely delaying progression from a specific disease, disorder, and / or condition; and / or reducing the risk of development of pathological aspects associated with a disease, disorder, and / or condition. In some aspects, the pharmaceutical formulations disclosed in this application may 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.

[0111] preventiveAs used herein, "preventive" refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay the symptoms of a disease or condition associated with fibrosis, e.g., NASH. In some aspects, the pharmaceutical formulations disclosed in this application may be used prophylactically.

[0112] prevention As used herein, "prevention" refers to measures taken to maintain health and to prevent or delay the onset of diseases or conditions associated with fibrosis, e.g., NASH or diabetes, or to prevent or delay symptoms associated with such diseases or conditions.

[0113] recombination "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, for the purposes of this disclosure, to be isolated, such as native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technology. The FGF-21 polypeptide disclosed herein may be recombinantly produced using methods known in the art. The proteins and peptides disclosed herein may also be chemically synthesized.

[0114] Similarity As used herein, the term "similarity" refers to the overall relationship between polymer molecules, for example, between polypeptide molecules. The calculation of the percentage similarity of polymer molecules to one another can be performed in the same manner as the calculation of percentage identity, except that the calculation of percentage similarity takes into account conservative substitutions as understood in the relevant art.

[0115] Solubility: The term “solubility” refers to the amount of a substance that can be dissolved in another substance, for example, the amount of unmodified or modified FGF-21 polypeptide that can be dissolved in an aqueous solution. An exemplary method for measuring the solubility of unmodified or modified FGF-21 polypeptide is the plug flow solubility test described in Example 8 of U.S. Application Publication No. US2017 / 0189486. For example, relative solubility with respect to a reference compound may be determined to identify polypeptides having increased solubility. In some aspects, for example, relative solubility with respect to a reference formulation may be determined to identify formulations in which polypeptides have increased solubility.

[0116] object : “Subject,” “individual,” “animal,” or “mammal” means any subject for whom diagnosis, prognosis, or therapy is desired, in particular a mammalian subject. Mammal subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sports animals, pets, e.g., dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates, e.g., apes, monkeys, orangutans, and chimpanzees; canids, e.g., dogs and wolves; felines, e.g., cats, lions, and tigers; equine animals, e.g., horses, donkeys, and zebras; bears; edible animals, e.g., cows, pigs, and sheep; ungulates, e.g., deer and giraffes; rodents, e.g., mice, rats, hamsters, and guinea pigs. In certain aspects, mammals are human subjects. In other aspects, subjects are human patients. In certain aspects, subjects are human patients or their cells applicable to the methods described herein in vivo, in vitro, or in vitro.

[0117] suffering: An individual “suffering” of a disease, disorder, and / or condition is one diagnosed with or exhibiting one or more symptoms of a disease, disorder, and / or condition. In some aspects, the pharmaceutical formulation disclosed herein may be administered to subjects suffering from a disease or condition associated with fibrosis, such as NASH or diabetes.

[0118] Easy to catch : Individuals "susceptible" to a disease, disorder, and / or condition may not be diagnosed with the disease, disorder, and / or condition or may not exhibit symptoms thereof, but have a tendency to develop the disease or its symptoms. In some aspects, individuals susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) genetic mutations associated with the development of the disease, disorder, and / or condition; (2) genetic polymorphisms associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition; (4) habits and / or lifestyle associated with the development 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 microorganisms associated with the development of the disease, disorder, and / or condition.

[0119] In some aspects, individuals susceptible to disease, disorder, and / or pathological condition will develop disease, disorder, and / or pathological condition. In some aspects, individuals susceptible to disease, disorder, and / or pathological condition will not develop disease, disorder, and / or pathological condition. In some aspects, the pharmaceutical formulation disclosed herein may be administered to subjects susceptible to diseases or pathological conditions associated with fibrosis, such as NASH or diabetes.

[0120] Therapeutic agents: The terms “therapeutic agent” or “agent” refer to a molecular entity that, when administered to a subject, has a therapeutic, diagnostic, and / or prophylactic effect or elicits a desired biological and / or pharmacological effect. For example, in some aspects, the FGF-21 polypeptide disclosed herein (e.g., PEG-FGF-21) may be a therapeutic agent. In some aspects, the agent is another molecule co-administered as part of a combination therapy with at least one of the FGF-21 polypeptides disclosed herein.

[0121] Therapeutic effective dose As used herein, the term “therapeutic effective dose” means an amount of agent (e.g., the FGF-21 polypeptide or preparations containing polypeptides disclosed herein) delivered that is sufficient to treat, / or alleviate, / or diagnose, / or prevent, or / or delay the onset of an infection, disease, disorder, and / or condition when administered to a subject suffering from or susceptible to such infection, disease, disorder, and / or condition. In general, administration of a therapeutic effective dose is expected to provide a therapeutically effective result.

[0122] Effective treatment results As used herein, the term "therapeutic effective result" means a result sufficient to treat, / or improve, / or diagnose, / or prevent, or / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to such infection, disease, disorder, and / or condition.

[0123] treat, treatment, therapyAs used herein, the terms “treat,” “therapy,” or “therapy,” or any grammatical variations thereof, refer to partially or completely preventing, alleviating, improving, and resolving, delaying the onset, inhibiting the progression, reducing the severity, or reducing the incidence of one or more symptoms or features of a disease or condition associated with fibrosis, e.g., NASH or diabetes. For example, “treating” a disease associated with fibrosis may refer to symptom prevention, symptom improvement, delaying the onset, etc., regarding the disease or condition or its symptoms. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition, and / or subjects who show only early signs of the disease, disorder, and / or condition, in order to reduce the risk of development of pathological aspects associated with the disease, disorder, and / or condition.

[0124] ug, uM, uL: As used herein, the terms "ug", "uM", and "uL" are used interchangeably with "μg", "μM", and "μL", respectively.

[0125] water-soluble polymer As used herein, the term “water-soluble polymer” refers to any polymer that is soluble in an aqueous solvent. The linkage of a water-soluble polymer to a modified FGF-21 polypeptide may provide changes including, but not limited to, an increased or modified serum (in vivo) half-life, or an increased or modified therapeutic half-life compared to the unmodified form, modified immunogenicity or toxicity, modified physical association characteristics, e.g., aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization.

[0126] A water-soluble polymer may or may not have biological activity of its own and may be utilized as a linker for the attachment of modified FGF-21 to one or more unmodified or modified FGF-21 polypeptides, or to other substances comprising, but not limited to, one or more biologically active molecules. Suitable polymers include polyethylene glycol, polyethylene glycol propionaldehyde, its mono C1-C10 alkoxy or aryloxy derivatives (described in U.S. Patent No. 5,252,714 incorporated herein by reference), monomethoxy-polyethylene glycol, distinct PEG, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives including but not limited to methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polypeptides, polyalkylene glycol and its derivatives, polyalkylene glycol and its derivatives. Copolymers, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, etc., or mixtures thereof, are included but not limited thereto. Examples of such water-soluble polymers include polyethylene glycol and serum albumin, but are not limited thereto.

[0127] Various aspects of the present disclosure are described in more detail in the following subsections.

[0128] I. FGF-21 Preparations

[0129] The present disclosure provides a pharmaceutical formulation having improved stability compared to a reference formulation, i.e., a corresponding formulation containing the same ingredients, except that it does not contain an aminopolycarboxylic acid cation chelator, comprising a fibroblast growth factor 21 (FGF-21) polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, and an aminopolycarboxylic acid cation chelator, e.g., diethylenetriaminepentaacetic acid (DTPA).

[0130] The terms “chelator” or “cation chelator” refer to any substance capable of removing a metal ion from a solution system by forming a new complex ion having chemical properties different from those of the original metal ion, which is interchangeable. In particular, the cation chelators disclosed herein are divalent metals, for example, Ca ++ It is a chelator that specifically binds to.

[0131] In some aspects, the pharmaceutical formulations disclosed herein exhibit one or more improvements in stability, e.g., a lower deamidation rate and / or a lower aggregation rate. For example, the inclusion of an aminopolycarboxylic acid cation chelator, e.g. DTPA, in the pharmaceutical formulation may reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g. PEG-FGF-21) when stored at a specific temperature (e.g., 40°C) for a specific time period (e.g., 1 month) with respect to the reference formulation.

[0132] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in pharmaceutical formulations stored at about 25°C, about 30°C, about 35°C, about 40°C, or about 45°C with respect to the reference formulation. In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in pharmaceutical formulations stored at temperatures above 25°C, above 30°C, above 35°C, about 40°C, or about 45°C with respect to the reference formulation.

[0133] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in pharmaceutical formulations stored at 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 with respect to the reference formulation.

[0134] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in a pharmaceutical formulation at the temperature or temperature range disclosed above 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 with respect to the reference formulation. In specific aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in a pharmaceutical formulation stored at 40°C for about 1 month with respect to the reference formulation.

[0135] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the aggregation rate of high molecular weight (HMW) FGF-21 polypeptides (e.g., modified FGF-21 polypeptides, e.g., PEG-FGF-21) in pharmaceutical formulations stored at about 25°C, about 30°C, about 35°C, about 40°C, or about 45°C with respect to the reference formulation. In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the aggregation rate of HMW FGF-21 polypeptides (e.g., modified FGF-21 polypeptides, e.g., PEG-FGF-21) in pharmaceutical formulations stored at temperatures above 25°C, above 30°C, above 35°C, about 40°C, or about 45°C with respect to the reference formulation.

[0136] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in pharmaceutical formulations stored at 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 with respect to the reference formulation.

[0137] In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in a pharmaceutical formulation at the temperature or temperature range disclosed above 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 with respect to the reference formulation. In specific aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the HMW aggregation rate of FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in a pharmaceutical formulation stored at 40°C for about 1 month with respect to the reference formulation.

[0138] In some aspects, an aminopolycarboxylic acid cation chelator, e.g., DTPA, prevents or reduces the oxidation of one or more amino acids, e.g., methionine, in an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21). In certain aspects, an aminopolycarboxylic acid cation chelator, e.g., DTPA, prevents or reduces the 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 or SEQ ID NO: 2 or any other FGF-21 polypeptide) at 25°C and / or 40°C.

[0139] In some specific aspects, the aminopolycarboxylic acid cation chelator is DTPA. Pentetate, or diethylenetriaminepentaacetic acid (DTPA), is an aminopolycarboxylic acid composed of a diethylenetriamine backbone having five carboxymethyl groups. The conjugate base of DTPA has a high affinity for metal cations. Therefore, the pentavalent anion DTPA 5- is, each nitrogen center and each COO - Assuming the group is counted as a center for coordination, it is a potential octate ligand. The formation constant for its complex is about 100 greater than that for EDTA. As a chelating agent, DTPA surrounds metal ions by forming up to eight bonds. However, transition metals typically form fewer than eight coordination bonds. Therefore, after forming a complex with a metal, DTPA still retains the ability to bind to other reagents, as indicated by its derivative pendettes. For example, in its complex with copper(II), DTPA binds in a hexate manner by utilizing three amine centers and three of the five carboxylates.

[0140] In some other aspect, the aminopolycarboxylic acid cation chelator may be another aminopolycarboxylic acid cation chelator, e.g., ethylenediamineacetic acid (EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,4,7,10-tetraazcyclododecane-1,4,7,10-tetraacetic acid (DOTA), or a related compound, e.g., thioxetane (a modified version of DTPA containing isothiocyanatobenzyl and methyl groups in its carbon backbone). Other chelating agents related to DTPA and EDTA known in the art have one or more C nitrogen groups in the amide group 1-18 Those that can be substituted by alkyl groups are, for example, DTPA.BMA and EDTA.BMA.

[0141] In some aspects, the DTPA cation chelator is present in amounts of about 10 μM to about 100 μM, 15 μM to about 95 μM, about 20 μM to about 90 μM, about 25 μM to about 85 μM, about 30 μM to about 80 μM, about 35 μM to about 75 μM, about 40 μM to about 70 μM, about 45 μM to about 65 μM, about 50 μM to about 60 μM, about 25 μM to about 75 μM, about 40 μM to about 60 μM, about 30 μM to about 70 μM, or about 40 μM to about 75 μM.

[0142] In some aspects, DTPA cation chelators are present in amounts 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.

[0143] In some aspects, the DTPA cation chelator is present in amounts 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.

[0144] Specifically, an aminopolycarboxylic acid cation chelator, for example, DTPA, is present in an amount of 50 μM.

[0145] In some aspects, 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 aspects, 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 aspects, the pH of the formulation is greater than about 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.

[0146] In some aspects, the pH of the formulation is 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 It is 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.

[0147] In some aspects, 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 aspects, the pharmaceutical formulation is more stable than the reference formulation having a pH of 6.5.

[0148] In some aspects, pharmaceutical formulations further comprise surfactants. As used herein, the term “surfactant” means any compound, typically an amphiphilic molecule, that reduces surface tension when dissolved or suspended in water or an aqueous solution, or reduces interfacial tension between two liquids or between a liquid and a solid. In the context of this disclosure, a surfactant is any compound that reduces interfacial stress and shear in a solution containing an FGF-21 polypeptide, for example, a modified FGF-21 polypeptide, such as PEG-FGF-21.

[0149] In some respects, surfactants are nonionic surfactants, that is, surfactants that tend not to have a net charge in neutral solutions. In some respects, non-anionic surfactants are polysorbates. Polysorbates are an important class of nonionic surfactants widely used in protein pharmaceuticals to stabilize proteins against interface-induced aggregation and to 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, nonionic surfactants composed of fatty acid esters of polyoxyethylene (POE) sorbitan. Commercially available polysorbates are chemically diverse mixtures containing mainly sorbitan POE fatty acid esters.

[0150] As used herein, the term “polysorbate” refers to oleate esters of sorbitol and its anhydride, typically copolymerized with ethylene oxide. Exemplary polysorbates are 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).

[0151] The number 20 following the 'polyoxyethylene' part refers to the total number of oxyethylene -(CH2CH2O)- groups present in the molecule. The number following the 'polysorbate' part concerns the type of fatty acid associated with the polyoxyethylene sorbitan portion of the molecule. Monolaurate is denoted by 20, monopalmitate by 40, monostearate by 60, and monooleate by 80. In some respects, non-ionic surfactants are present in amounts exceeding the critical micelle concentration (CMC), which is approximately at least 0.01 mg / ml in the case of polyoxyethylene sorbitan fatty acid esters. See reference [Wan and Lee, Journal of Pharm Sci, 63, p.136, 1974].

[0152] In some respects, polysorbate is polysorbate 80 (PS80). In some aspects, polysorbate 80 surfactants are 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 It is present in an amount of 0.05% to about 0.06% (w / v).

[0153] In some aspects, polysorbate 80 surfactant 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).

[0154] In some aspects, a surfactant, e.g., polysorbate 80, reduces particulate and / or bubble formation when stirring the formulation, e.g., on a shaker. In some aspects, the presence of a surfactant, e.g., polysorbate 80, in the formulation can 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 level of particulate formation in the reference formulation. In some aspects, the presence of a surfactant in the formulation, for example, polysorbate 80, can reduce foam 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 level of foam formation in the reference formulation.

[0155] In some aspects, pharmaceutical formulations additionally contain amino acid buffers. Amino acids can be advantageously used as buffers in pharmaceutical applications because they are naturally occurring substances that are easily metabolizable. Additionally, amino acids used as buffers can also protect proteins in the amorphous phase when the formulation is freeze-dried. Suitable amino acid buffers may contain histidine, lysine, and / or arginine. Histidine has excellent buffering capacity at approximately pH 7.

[0156] As used herein, the term “histidine” includes L-histidine or D-histidine, the solvated form of histidine, the hydrated form of histidine (e.g., monohydrate), or the anhydrous form of histidine, or a mixture thereof. Other suitable buffers in the formulations of this disclosure are, to name only a few, glutamate, Tris, or succinate.

[0157] In some specific aspects, the amino acid buffer is L-histidine.

[0158] In some aspects, the histidine buffer comprises 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, 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 It is present in amounts of 85 mM to about 100 mM histidine, about 90 mM to about 100 mM histidine, or about 95 mM to about 100 mM histidine.

[0159] In some aspects, the histidine buffer comprises 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 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, and about 35 mM to about 80 mM Histidine is present in amounts of about 40 mM to about 80 mM histidine, and about 45 mM to about 80 mM histidine.

[0160] In some aspects, the histidine buffer comprises 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, and about 25 mM to about 30 mM Histidine is present in amounts of about 15 mM to about 25 mM histidine, or about 20 mM to about 25 mM.

[0161] In some aspects, histidine buffer is present in amounts 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.

[0162] In some aspects, the histidine buffer contains approximately 10 mM histidine, approximately 11 mM histidine, approximately 12 mM histidine, approximately 13 mM histidine, approximately 14 mM histidine, approximately 15 mM histidine, approximately 16 mM histidine, approximately 17 mM histidine, approximately 18 mM histidine, approximately 19 mM histidine, approximately 20 mM histidine, approximately 21 mM histidine, approximately 22 mM histidine, approximately 23 mM histidine, approximately 24 mM histidine, approximately 25 mM histidine, approximately 26 mM histidine, approximately 27 mM histidine, approximately 28 mM histidine, approximately 29 mM histidine, approximately 30 mM histidine, approximately 31 mM histidine, approximately 32 mM histidine, approximately 33 mM histidine, approximately 34 mM histidine, approximately 35 mM histidine, and approximately 36 It is present in amounts of 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.

[0163] In some aspects, the pharmaceutical formulation further comprises an osmotic regulator (also known in the art as an isotonic agent). According to the present disclosure, the osmotic regulator (isotonic agent) may comprise a polyol, a saccharide, a carbohydrate, a salt, such as sodium chloride, or a mixture thereof. Exemplary polyols include those having a molecular weight of less than about 600 kD (e.g., in the range of 120 to 400 kD), such as mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or a mixture thereof.

[0164] Saccharide or carbohydrate osmotic regulators include monosaccharides, disaccharides and polysaccharides, or mixtures thereof. In some aspects, the saccharide 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.

[0165] In some aspects, the osmotic regulator comprises a saccharide selected from the group of reducing sugars or non-reducing sugars or mixtures thereof. In some aspects, the osmotic regulator isotonic agent comprises a sugar selected from the group consisting of non-reducing sugars, preferably sucrose, trehalose, and mixtures thereof. In some specific aspects, the non-reducing sugar is sucrose.

[0166] In some aspects, sucrose osmotic regulators are 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, about 600 mM to about 900 mM sucrose, about 700 mM to about 900 mM It is present in amounts of 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.

[0167] In some aspects, sucrose osmotic regulators are present in amounts 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.

[0168] In some aspects of the pharmaceutical formulations disclosed herein, the FGF-21 polypeptide is a modified FGF-21 polypeptide. Many modified FGF-21 polypeptides known in the art, such as those disclosed in U.S. Patent Nos. 8,012,931 and 9,434,788, the full text of which is incorporated herein by reference, may be used in the formulations disclosed herein. Fibroblast growth factor 21 (FGF-21) is described in the literature (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206 (2000); WO 01 / 36640; and WO 01 / 18172, and U.S. Patent Publication No. 20040259780, each of which is incorporated herein by reference in its full text). Unlike other FGFs, FGF-21 has been reported not to have proliferative and tumorigenic effects (Ornitz and Itoh, Genome Biology 2001, 2(3):reviews3005.1-3005.12).

[0169] Specific FGF-21 polypeptides and their uses are U.S. Patent Publication No. 20010012628, U.S. Patent No. 6,716,626, U.S. Patent Publication No. 2004 / 0259780, WO 03 / 011213, Kharitonenkov et al. J Clin Invest. 2005 June; 115(6): 1627-35, WO 03 / 059270, U.S. Patent Publication No. 2005 / 0176631, WO 2005 / 091944, WO 2007 / 0293430, U.S. Patent Publication No. 2007 / 0293430, WO / 2008 / 121563, U.S. Patent No. 4,904,584, WO 99 / 67291, WO 99 / 03887, WO 00 / 26354, and U.S. Patent No. 5,218,092, each of which is incorporated herein by reference in its entirety.

[0170] Additional FGF-21 that can be formulated as disclosed in this application is described in more detail below.

[0171] In some aspects, the modified FGF-21 polypeptide comprises 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 with respect to the amino acid sequence of sequence identification number: 1 or sequence identification number: 3, wherein the polypeptide is FGF-21 active.

[0172] In some aspects, the modified FGF-21 polypeptide is composed 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 with respect to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or is essentially composed of such a polypeptide, wherein the polypeptide has FGF-21 activity.

[0173] In some aspects, the modified FGF-21 polypeptide is linked to a half-life extension moiety, for example, through a side chain of non-naturally encoded aminos within the sequence of the FGF-21 polypeptide. Half-life extension moietyes that may be linked to the modified FGF-21 polypeptide of the present disclosure, for example, the FGF-21 polypeptide of SEQ ID NO: 1, include, for example, albumin, an immunoglobulin constant region or part thereof, an immunoglobulin-binding polypeptide, immunoglobulin G (IgG), an albumin-binding polypeptide (ABP), a PAS-modified moiety, an HES-modified moiety, an XTEN, an Fc region, and any combination thereof. Half-life extension moietyes that may be used to modify the FGF-21 polypeptide are described in more detail below.

[0174] In some aspects, the half-life extension moiety comprises a water-soluble polymer, e.g., polyethylene glycol (PEG). In some aspects, PEG has an average molecular weight of about 10 kDa to about 40 kDa. Thus, in some aspects, PEG has an average molecular weight of about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa.

[0175] In some aspects, PEG has an average molecular weight of about 30 kDa. In some aspects, PEG has an average molecular weight of about 15 kDa to about 40 kDa, about 20 kDa to about 40 kDa, about 25 kDa to about 40 kDa, about 30 kDa to about 40 kDa, about 35 kDa to about 40 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 35 kDa, about 25 kDa to about 35 kDa, or about 30 kDa to about 35 kDa.

[0176] In some aspects, a half-life extension moiety, e.g., PEG, is linked to the FGF-21 polypeptide (e.g., the modified FGF-21 polypeptide of SEQ ID No. 1 or SEQ ID No. 2) via a non-naturally coded amino acid, e.g., a phenylalanine derivative. In some aspects, the phenylalanine derivative is para-acetyl-L-phenylalanine. In some aspects, the half-life extension moiety is linked to the non-naturally coded amino acid via an oxime linkage. In some aspects, the non-naturally coded amino acid replaces the amino acid glutamine 109 of SEQ ID No. 3 (wild-type FGF-21).

[0177] In some specific aspects of the present disclosure, the FGF-21 polypeptide is a derivative of the modified FGF-21 of SEQ ID NO: 1, wherein PEG-FGF-21 (SEQ ID NO: 2), i.e., glutamine 109 of wild-type FGF-21 (SEQ ID NO: 3) is replaced with para-acetyl-L-phenylalanine and a 30 kDa PEG chain is covalently attached to para-acetyl-L-phenylalanine at position 109 via an oxime linkage.

[0178] In some aspects, FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) is present at a concentration of about 1 mg / ml to about 40 mg / ml. In some aspects, FGF-21 polypeptide 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 aspects, FGF-21 polypeptides (e.g., modified FGF-21 polypeptides, e.g., PEG-FGF-21) are present at concentrations of about 1 mg / ml to 5 mg / ml, about 5 mg / ml to about 10 mg / ml, about 10 mg / ml to about 15 mg / ml, about 15 mg / ml to about 20 mg / ml, about 20 mg / ml to about 25 mg / ml, about 25 mg / ml to about 30 mg / ml, about 30 mg / ml to about 35 mg / ml, or about 35 mg / ml to about 40 mg / ml.

[0179] In some aspects, FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) is 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, It is present at concentrations of 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, or 40 mg / ml. In some aspects, the concentration of FGF-21 polypeptide is determined according to methods known in the art, or specific methods disclosed in the examples section of this application.

[0180] In some aspects, FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) is present in the formulations disclosed herein in an amount of about 1 mg to about 40 mg per dose. In some aspects, FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) is present in the formulations disclosed herein in an amount of about 1 mg to about 5 mg per dose, about 5 mg to about 10 mg per dose, or about 10 mg to about 15 mg per dose, about 15 mg to about 20 mg per dose, or about 20 mg to about 25 mg per dose, about 25 mg to about 30 mg per dose, or about 30 mg to about 35 mg per dose, or about 35 mg to about 40 mg per dose. In some aspects, FGF-21 polypeptide, for example, modified FGF-21 polypeptide, e.g., PEG-FGF-21, is present in the formulations disclosed herein in amounts exceeding 40 mg per dose.

[0181] In some aspects, FGF-21 polypeptide (e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21) in the formulations disclosed herein is 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 It is present in amounts of 27 mg, 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, or about 40 mg per dose.

[0182] In some aspects, pharmaceutical formulations are formulated for subcutaneous administration. As discussed below, other pharmaceutical formulations disclosed herein may be administered via other routes. In some aspects, pharmaceutical formulations are formulated for subcutaneous administration, for example, via a safety syringe. In some aspects, formulations are formulated for daily or weekly administration, for example, every 1, 2, 3, 4, 5, or 6 days, weekly, or every two weeks. In some aspects, formulations are aqueous formulations.

[0183] In some aspects, the present disclosure provides a pharmaceutical formulation comprising: (i) an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21; (ii) histidine at a concentration of about 10 mM to about 50 mM; (iii) sucrose at a concentration of about 100 mM to about 1 M; (iv) polysorbate 80 at a concentration of about 0.01% to about 0.1% (w / v); and (v) DTPA at a concentration of about 10 μM to about 100 μM; wherein the pH of the formulation is about 6.7 to about 7.5.

[0184] In addition, a pharmaceutical formulation comprising the following is provided: (i) an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21; (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 pH is about 7.1.

[0185] In addition, a pharmaceutical formulation comprising the following is provided: (i) FGF-21 polypeptide, e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21; (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.

[0186] In addition, a pharmaceutical formulation is provided comprising: (i) an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21; (ii) histidine at a concentration of about 20 mM; and (iii) sucrose at a concentration of about 600 mM; wherein the pH is about 7.0.

[0187] In addition, a pharmaceutical formulation comprising the following is provided: (i) an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21; (ii) histidine at a concentration of 20 mM; and (iii) sucrose at a concentration of 600 mM; wherein the pH is 7.0.

[0188] In some specific aspects, the present disclosure provides a pharmaceutical formulation comprising: (i) PEG-FGF-21 (sequence identification number: 2) 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.

[0189] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF-21 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 pH is about 7.1.

[0190] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF-21 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.

[0191] In addition, a pharmaceutical formulation comprising the following is provided: (i) PEG-FGF21 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.

[0192] The present disclosure also provides a pharmaceutical formulation prepared according to any of the methods for improving the stability of a formulation comprising FGF-21 (e.g., modified FGF-21, e.g., PEG-FGF-21) disclosed below.

[0193] II. Manufacturing Method

[0194] The present disclosure also provides a method for improving or enhancing the stability of a pharmaceutical formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing an aminopolycarboxylic acid cation chelator, e.g., DTPA, wherein the formulation has improved stability compared to a reference formulation not containing an aminopolycarboxylic acid cation chelator. These stabilization methods include (i) mixing the aminopolycarboxylic acid cation chelator into the formulation, (ii) mixing a polysorbate surfactant, e.g., polysorbate 80, (iii) adjusting the pH of the formulation to approximately 7.1, or (iv) any combination thereof.

[0195] In some aspects, the pharmaceutical formulation described herein is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of about 10 mg / mL; (ii) an amount of histidine to achieve a final concentration of about 20 mM; (iii) an amount of sucrose to achieve a final concentration of about 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of about 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of about 50 uM; and adjusting the pH to about 7.1.

[0196] In some aspects, the pharmaceutical formulation described herein is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of about 20 mg / mL; (ii) an amount of histidine to achieve a final concentration of about 20 mM; (iii) an amount of sucrose to achieve a final concentration of about 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of about 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of about 50 μM; and adjusting the pH to about 7.1.

[0197] In some aspects, the pharmaceutical formulation described herein is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of 10 mg / mL; (ii) an amount of histidine to achieve a final concentration of 20 mM; (iii) an amount of sucrose to achieve a final concentration of 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of 50 uM; and adjusting the pH to 7.1.

[0198] In some aspects, the pharmaceutical formulation described herein is prepared by a process of mixing (i) an amount of PEG-FGF-21 to achieve a final concentration of 20 mg / mL; (ii) an amount of histidine to achieve a final concentration of 20 mM; (iii) an amount of sucrose to achieve a final concentration of 600 mM; (iv) an amount of polysorbate 80 to achieve a final concentration of 0.05% (w / v); and (v) an amount of DTPA to achieve a final concentration of 50 uM; and adjusting the pH to 7.1.

[0199] As used herein, the term “mixing” refers to combining the components of the formulation disclosed herein in no predetermined order to reach a disclosed concentration by any means known in the relevant art. For example, excipients in the pharmaceutical formulation disclosed herein may be added to the FGF-21 polypeptide solution sequentially or simultaneously. Alternatively, a concentrated FGF-21 polypeptide solution may be added to a solution containing all or part of the excipients in the formulation. In another aspect, the excipients may be incorporated into the formulation, for example, by dialysis or filtration.

[0200] In some aspects, improvements in stability resulting from the application of the disclosed method include, for example, (i) an increase in physical stability with respect to the reference formulation, (ii) an increase in chemical stability with respect to the reference formulation, or (iii) a combination thereof.

[0201] In some aspects, an increase in physical stability comprises (i) prevention or reduction of polypeptide aggregation, (ii) prevention or reduction of polypeptide fragmentation, or (iii) a combination thereof. In some aspects, polypeptide aggregation 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 aggregation observed in the reference formulations. In some aspects, polypeptide aggregation observed in the 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 aggregation observed in the reference formulation. In some aspects, polypeptide fragmentation observed in the 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 fragmentation appearing in the reference formulation. In some aspects, polypeptide fragmentation observed in the 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 fragmentation observed in the reference formulation.

[0202] In some aspects, an increase in chemical stability comprises (i) prevention or reduction of polypeptide deamidation, (ii) prevention or reduction of polypeptide oxidation, or (iii) a combination thereof. In some aspects, polypeptide deamidation 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 polypeptide deamidation observed in the reference formulations. In some aspects, polypeptide deamidation observed in the 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 polypeptide deamidation observed in the reference formulation. In some aspects, polypeptide oxidation observed in the 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 polypeptide oxidation observed in the reference formulation. In some aspects, polypeptide oxidation observed in the 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 polypeptide oxidation observed in the reference formulation.

[0203] In some aspects, a method for stabilizing (improving or enhancing stability) the formulation disclosed herein may reduce the rate of degradation as well as the level of degradation: for example, the disclosed method may, with respect to the reference formulation, reduce the rate of polypeptide deamidation, reduce the rate of polypeptide oxidation, reduce the rate of polypeptide aggregation, reduce the rate of proteolytic degradation, or any combination thereof.

[0204] For example, the inclusion of an aminopolycarboxylic acid cation chelator, such as DTPA, in the pharmaceutical formulation disclosed herein can reduce the deamidation rate of FGF-21 polypeptide, for example, modified FGF-21 polypeptide, for example, PEG-FGF-21, when stored at a specific temperature for a specific period of time with respect to the reference formulation.

[0205] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the deamidation rate of the FGF-21 polypeptide with respect to the 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. In some aspects, the aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of the FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, with respect to the reference formulation when the pharmaceutical formulation is stored at temperatures above 25°C, above 30°C, above 35°C, about 40°C, or about 45°C. In some aspects, an aminopolycarboxylic acid cation chelator (e.g., DTPA) can reduce the deamidation rate of FGF-21 polypeptide, e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21, when the pharmaceutical formulation is stored at 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 with respect to the reference formulation.

[0206] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the rate of deamidation of the FGF-21 polypeptide when the pharmaceutical formulation is stored at the 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 with respect to the reference formulation.

[0207] In a specific aspect, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the rate of deamidation of the FGF-21 polypeptide when the pharmaceutical formulation is stored at 40°C for about one month with respect to the reference formulation.

[0208] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the aggregation rate of the high molecular weight (HMW) FGF-21 polypeptide when the pharmaceutical formulation is stored at about 25°C, about 30°C, about 35°C, about 40°C, or about 45°C for the reference formulation.

[0209] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the HMW aggregation rate of the FGF-21 polypeptide when the pharmaceutical formulation is stored at temperatures above 25°C, above 30°C, above 35°C, about 40°C, or about 45°C for reference formulations. In some aspects, mixing an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the HMW aggregation rate of the FGF-21 polypeptide when the pharmaceutical formulation is stored at a temperature of 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 for the reference formulation.

[0210] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the HMW aggregation rate of the FGF-21 polypeptide when the pharmaceutical formulation is stored at the 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 with respect to the reference formulation.

[0211] In a specific aspect, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can reduce the HMW aggregation rate of the FGF-21 polypeptide when the pharmaceutical formulation is stored at 40°C for about one month with respect to the reference formulation.

[0212] In some aspects, the mixing of an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can prevent or reduce the oxidation of one or more methionines within the FGF-21 polypeptide.

[0213] In certain aspects, mixing an aminopolycarboxylic acid cation chelator (e.g., DTPA) with a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) can prevent or reduce the 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 any other FGF-21 polypeptide) at, for example, 25°C and / or 40°C.

[0214] The disclosed method may use the aminopolycarboxylic acid cation chelator DTPA. However, in some other aspects, the aminopolycarboxylic acid cation chelator may be, for example, another aminopolycarboxylic acid cation chelator, such as EDTA, EGTA, DOTA, DTPA-related compounds, such as thioxetane, or any chelating agent related to DTPA and EDTA known in the art, such as DTPA.BMA and EDTA.BMA.

[0215] In some aspects, the DTPA cation chelator may be mixed in amounts of about 10 μM to about 100 μM, 15 μM to about 95 μM, about 20 μM to about 90 μM, about 25 μM to about 85 μM, about 30 μM to about 80 μM, about 35 μM to about 75 μM, about 40 μM to about 70 μM, about 45 μM to about 65 μM, about 50 μM to about 60 μM, about 25 μM to about 75 μM, about 40 μM to about 60 μM, about 30 μM to about 70 μM, or about 40 μM to about 75 μM.

[0216] In some aspects, the DTPA cation chelator can be mixed in amounts 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.

[0217] In some aspects, the DTPA cation chelator may be mixed 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.

[0218] Specifically, an aminopolycarboxylic acid cation chelator, for example, DTPA, is mixed in an amount of 50 μM.

[0219] In some aspects of the method 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.

[0220] In some aspects, 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.

[0221] In some aspects, 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.

[0222] In some aspects, the pH of the formulation is 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 It is adjusted to a pH of 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.

[0223] In some aspects, 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 aspects, the pharmaceutical formulation is more stable than the reference formulation having a pH of 6.5 after pH adjustment.

[0224] In some aspects, 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 aspects, the pharmaceutical formulation is more stable than the reference formulation having a pH of 6.5 after pH adjustment.

[0225] In some aspects, a method for improving the stability of a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) further comprises mixing a surfactant. In some aspects, the mixed surfactant is a nonionic surfactant, that is, a surfactant that tends not to have a net charge in a neutral solution. In some aspects, the mixed non-anionic surfactant is a polysorbate. In some aspects, the non-ionic surfactant is mixed in an amount exceeding the critical micelle concentration (CMC), which is approximately at least 0.01 mg / ml in the case of polyoxyethylene sorbitan fatty acid ester. See reference [Wan and Lee, Journal of Pharm Sci, 63, p.136, 1974]. In some aspects of the method of the present invention, the polysorbate is polysorbate 80 (PS80).

[0226] In some aspects, the PS80 surfactant 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 It is mixed into pharmaceutical formulations in an amount of 0.05% to about 0.06% (w / v).

[0227] In some aspects, the polysorbate 80 surfactant 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 aspects, the surfactant, e.g., PS80, is mixed in an amount sufficient to reduce or prevent particulate formation and / or bubble formation, e.g., when stirring the formulation (e.g., on a shaker).

[0228] In some aspects, a method for improving the stability of a pharmaceutical formulation containing an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) further comprises mixing an amino acid buffer, e.g., histidine (i.e., L-histidine, D-histidine, solvated histidine, hydrated histidine, anhydrous histidine, or a mixture thereof).

[0229] In some aspects, 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 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, 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 histidine, about 15 mM to about 25 mM histidine, about 15 mM to about 25 mM, about 15 mM to about It is mixed in amounts of 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.

[0230] In some aspects, the histidine buffer contains approximately 10 mM histidine, approximately 11 mM histidine, approximately 12 mM histidine, approximately 13 mM histidine, approximately 14 mM histidine, approximately 15 mM histidine, approximately 16 mM histidine, approximately 17 mM histidine, approximately 18 mM histidine, approximately 19 mM histidine, approximately 20 mM histidine, approximately 21 mM histidine, approximately 22 mM histidine, approximately 23 mM histidine, approximately 24 mM histidine, approximately 25 mM histidine, approximately 26 mM histidine, approximately 27 mM histidine, approximately 28 mM histidine, approximately 29 mM histidine, approximately 30 mM histidine, approximately 31 mM histidine, approximately 32 mM histidine, approximately 33 mM histidine, approximately 34 mM histidine, approximately 35 mM histidine, and approximately 36 It is mixed in amounts of 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.

[0231] In some aspects, a method for improving the stability of a pharmaceutical formulation comprising an FGF-21 polypeptide (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) further comprises mixing an osmotic regulator (isotonic agent). According to the present disclosure, the osmotic regulator (isotonic agent) may comprise a polyol, a saccharide, a carbohydrate, a salt, e.g. sodium chloride, or a mixture thereof. Exemplary polyols include those having a molecular weight of less than about 600 kD (e.g., in the range of 120 to 400 kD), e.g. mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, or a mixture thereof. Saccharide or carbohydrate osmotic regulators include monosaccharides, disaccharides, and polysaccharides, or mixtures thereof. In some aspects, the saccharide 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 regulator comprises a saccharide selected from the group consisting of reducing sugars or non-reducing sugars or mixtures thereof. In some aspects, the osmotic regulator isotonic agent comprises a saccharide that is a non-reducing sugar, preferably selected from the group consisting of sucrose, trehalose, and mixtures thereof. In some specific aspects, the non-reducing sugar is sucrose.

[0232] In some aspects, sucrose osmotic regulators are 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, about 600 mM to about 900 mM sucrose, about 700 mM to about 900 mM It is mixed in amounts of 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.

[0233] In some aspects, sucrose osmotic regulators are mixed in amounts 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.

[0234] In some aspects, the FGF-21 polypeptide is a modified FGF-21 polypeptide. Many modified FGF-21 polypeptides known in the art, such as those disclosed in U.S. Patent Nos. 8,012,931 and 9,434,788, the full text of which is incorporated herein by reference, may be used in the methods disclosed herein. Additional FGF-21s that may be formulated as disclosed in this application are described in more detail below.

[0235] In some aspects, the modified FGF-21 polypeptide useful in the method disclosed herein comprises 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 with respect to the amino acid sequence of sequence identification number: 3 (wild-type humFGF-21), wherein the polypeptide has FGF-21 activity.

[0236] In some aspects, the modified FGF-21 polypeptide useful in the method disclosed herein is composed of or essentially composed 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 with respect to the amino acid sequence of sequence identification number: 3 (wild type humFGF-21), wherein the polypeptide has FGF-21 activity.

[0237] In some aspects, the modified FGF-21 polypeptide useful in the method disclosed herein is linked to a half-life extension moiety, for example, through a side chain of non-naturally encoded aminos within the sequence of the FGF-21 polypeptide. Half-life extension moietyes that can be linked to the FGF-21 polypeptide of the disclosure, for example, the modified FGF-21 polypeptide of SEQ ID NO: 1, include, for example, albumin, an immunoglobulin constant region or part thereof, an immunoglobulin-binding polypeptide, IgG, ABP, a PAS-modified moiety, an HES-modified moiety, XTEN, an Fc region, and any combination thereof. Half-life extension moietyes that can be used to modify the FGF-21 polypeptide are described in more detail below.

[0238] In some aspects, the half-life extending moiety useful in the method disclosed herein comprises a water-soluble polymer, e.g., polyethylene glycol (PEG). In some aspects, PEG has an average molecular weight of about 10 kDa to about 40 kDa, e.g., about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, or about 40 kDa. In some aspects, PEG has an average molecular weight of about 30 kDa. In some aspects, PEG has an average molecular weight of about 15 kDa to about 40 kDa, about 20 kDa to about 40 kDa, about 25 kDa to about 40 kDa, about 30 kDa to about 40 kDa, about 35 kDa to about 40 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 20 kDa, about 20 kDa to about 35 kDa, about 25 kDa to about 35 kDa, or 30 kDa to about 35 kDa.

[0239] In some aspects, a half-life extension moiety, e.g., PEG, is linked to the FGF-21 polypeptide via a non-naturally coded amino acid, e.g., a phenylalanine derivative. In some aspects, the phenylalanine derivative is para-acetyl-L-phenylalanine. In some aspects, the half-life extension moiety is linked to the non-naturally coded amino acid via an oxime linkage. In some aspects, the non-naturally coded amino acid replaces the amino acid glutamine 109 of SEQ ID NO: 3. In some aspects, the FGF-21 polypeptide is PEG-FGF-21 (SEQ ID NO: 2), i.e., the FGF-21 of SEQ ID NO: 1, in which glutamine 109 is replaced by para-acetyl-L-phenylalanine and a 30 kDa PEG chain is covalently attached to para-acetyl-L-phenylalanine via an oxime linkage.

[0240] In some aspects of the method disclosed herein, the FGF-21 polypeptide is present at a concentration of about 1 mg / ml to about 40 mg / ml. In some aspects, the FGF-21 polypeptide 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 aspects, the concentration of the FGF-21 polypeptide is determined according to a method known in the art or a specific method disclosed in the Examples section of this application.

[0241] In some aspects of the method 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.

[0242] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing (i) histidine at a concentration of about 10 mM to about 50 mM; (ii) sucrose at a concentration of about 100 mM to about 1 M; (iii) polysorbate 80 at a concentration of about 0.01% to about 0.1% (w / v); and (iv) DTPA at a concentration of about 10 μM to about 100 μM; wherein the pH of the formulation is about 6.7 to about 7.5.

[0243] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing (i) histidine at a concentration of about 15 mM to about 45 mM; (ii) sucrose at a concentration of about 200 mM to about 900 mM; (iii) polysorbate 80 at a concentration of about 0.02% to about 0.09% (w / v); and (iv) DTPA at a concentration of about 20 μM to about 90 μM; wherein the pH of the formulation is about 6.8 to about 7.4.

[0244] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing (i) histidine at a concentration of about 15 mM to about 40 mM; (ii) sucrose at a concentration of about 300 mM to about 800 mM; (iii) polysorbate 80 at a concentration of about 0.03% to about 0.08% (w / v); and (iv) DTPA at a concentration of about 30 μM to about 80 μM; wherein the pH of the formulation is about 6.9 to about 7.3.

[0245] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing (i) histidine at a concentration of about 15 mM to about 30 mM; (ii) sucrose at a concentration of about 400 mM to about 800 mM; (iii) polysorbate 80 at a concentration of about 0.04% to about 0.07% (w / v); and (iv) DTPA at a concentration of about 40 μM to about 70 μM; wherein the pH of the formulation is about 7 to about 7.2.

[0246] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, comprising mixing (i) histidine at a concentration of about 15 mM to about 25 mM; (ii) sucrose at a concentration of about 500 mM to about 700 mM; (iii) polysorbate 80 at a concentration of about 0.04% to about 0.06% (w / v); and (iv) DTPA at a concentration of about 45 μM to about 55 μM; wherein the pH of the formulation is about 7 to about 7.1.

[0247] The present disclosure also provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, 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.

[0248] The present disclosure also provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, 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.

[0249] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, 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.

[0250] The present disclosure provides a method for improving the stability of a formulation comprising an FGF-21 polypeptide, e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21, 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.

[0251] The present disclosure provides a method for improving the stability of a formulation containing PEG-FGF-21 (sequence identification number: 2) 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 μM; wherein the pH is about 7.1.

[0252] The present disclosure provides a method for improving the stability of a formulation containing PEG-FGF-21 (sequence identification number: 2) 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.

[0253] The present disclosure provides a method for improving the stability of a formulation containing PEG-FGF-21 (sequence identification number: 2) 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.

[0254] The present disclosure provides a method for improving the stability of a formulation containing PEG-FGF-21 (sequence identification number: 2) 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.

[0255] In some embodiments, the concentration of individual excipients (e.g., DTPA, PS80, histidine, sucrose) included in the pharmaceutical formulation may be determined / calculated as the amount (weight, mole, etc.) of the individual excipient added to the pharmaceutical formulation during the manufacturing process per unit of the final volume of the finished pharmaceutical formulation. In other embodiments, the concentration of excipients (e.g., DTPA, PS80, histidine, sucrose) included in the pharmaceutical formulation is based on the actual amount of the individual excipient in the pharmaceutical formulation.

[0256] III. Treatment Methods

[0257] The present disclosure also provides a method for treating or preventing a disease or condition associated with fibrosis and / or diabetes in a subject who requires treatment or prevention of a disease or condition associated with fibrosis and / or diabetes, comprising administering an effective amount of a pharmaceutical preparation containing FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21) to the subject who requires treatment or prevention of a disease or condition associated with fibrosis and / or diabetes.

[0258] In some aspects, the disease or condition is diabetes, for example, type 2 diabetes. In some aspects, the disease or condition is non-alcoholic fatty liver disease (NASH).

[0259] In some aspects, FGF-21 polypeptides, for example, modified FGF-21 polypeptides, such as PEG-FGF-21, may be used in the treatment or prevention methods disclosed herein in an amount of about 1 mg to about 40 mg per dose. In some aspects, FGF-21 polypeptides, for example, modified FGF-21 polypeptides, such as PEG-FGF-21, may be used in the treatment or prevention methods disclosed herein in an amount of about 1 mg to about 5 mg per dose, or about 5 mg to about 10 mg per dose, or about 10 mg to about 15 mg per dose, or about 15 mg to about 20 mg per dose, or about 20 mg to about 25 mg per dose, or about 25 mg to about 30 mg per dose, or about 30 mg to about 35 mg per dose, or about 35 mg to about 40 mg per dose. In some aspects, FGF-21 polypeptides, for example, modified FGF-21 polypeptides, such as PEG-FGF-21, may be used in the treatment or prevention methods disclosed herein in amounts exceeding 40 mg per dose.

[0260] In some aspects, FGF-21 polypeptide, e.g., modified FGF-21 polypeptide, e.g., PEG-FGF-21, per dose approximately 1 mg, approximately 2 mg, approximately 3 mg, approximately 4 mg, approximately 5 mg, approximately 6 mg, approximately 7 mg, approximately 8 mg, approximately 9 mg, approximately 10 mg, approximately 11 mg, approximately 12 mg, approximately 13 mg, approximately 14 mg, approximately 15 mg, approximately 16 mg, approximately 17 mg, approximately 18 mg, approximately 19 mg, approximately 20 mg, approximately 21 mg, approximately 22 mg, approximately 23 mg, approximately 24 mg, approximately 25 mg, approximately 26 mg, approximately 27 mg, approximately 28 mg, approximately 29 mg, approximately 30 mg, approximately 31 mg, approximately 32 mg, approximately 33 mg, approximately 34 mg, approximately 35 mg, approximately 36 mg, It may be used in the treatment or prevention methods disclosed herein in amounts of about 37 mg, about 38 mg, about 39 mg, or about 40 mg.

[0261] In some aspects, pharmaceutical preparations are administered subcutaneously, for example, using a safety syringe. In some aspects, pharmaceutical preparations are administered daily or weekly.

[0262] In some aspects, administration of a pharmaceutical formulation containing an effective amount of the FGF-21 disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21) to a subject reduces liver stiffness, reduces body fat percentage, reduces body weight, reduces liver-to-body-replacement 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.

[0263] In some aspects, administration of a pharmaceutical formulation comprising the FGF-21 polypeptide disclosed herein (e.g., a modified FGF-21 polypeptide, e.g., PEG-FGF-21) according to the treatment method disclosed herein to a subject results in, compared to levels in an untreated subject or to a subject prior to administration of the pharmaceutical formulation, (i) a decrease in the level of hepatic steatosis; (ii) a decrease in the level of hepatic impairment; (iii) a decrease in the level of fibrosis; (iv) a decrease in the level of the fibrosis biomarker serum Pro-C3 (N-terminal type III collagen propeptide); (v) a decrease in the level of alanine aminotransferase (ALT); (vi) a decrease in the level of aspartate aminotransferase (AST); (vii) an increase in the level of serum adiponectin; (viii) a decrease in the level of plasma LDL; (ix) an increase in the level of plasma HDL; (x) a decrease in the level of plasma triglycerides; (xi) reduction in the level of liver stiffness; or (xii) any combination of these.

[0264] The present disclosure provides a method for treating a fibrosis-associated disease, comprising administering an effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide disclosed herein to a subject requiring treatment for a fibrosis-associated disease. In some aspects, the fibrosis-associated disease may affect organs and tissues such as the pancreas, lungs, heart, kidneys, liver, eyes, nervous system, bone marrow, lymph nodes, endocardial myocardium, and / or retroperitoneal space. In some aspects, the fibrosis-associated disease may be hepatic fibrosis or pre-cirrhosis. In some aspects, diseases associated with fibrosis may be selected from the following: non-alcoholic steatohepatitis (NASH), cirrhosis, diffuse parenchymal lung disease, cystic fibrosis, pulmonary fibrosis, progressive macrofibrosis, idiopathic pulmonary fibrosis, infusion fibrosis, renal fibrosis, chronic kidney disease, diabetic nephropathy, focal segmental glomerulosclerosis, membranous nephropathy, IgA nephropathy, myelofibrosis, heart failure, acute heart failure, chronic heart failure, metabolic heart failure, cardiac fibrosis, cataract fibrosis, cataract, ocular scarring, pancreatic fibrosis, cutaneous fibrosis, intestinal fibrosis, intestinal stenosis, endocardial cardiomyopathy, atrial fibrosis, mediastinal fibrosis, Crohn's disease, retroperitoneal fibrosis, keloid, nephrogenic systemic fibrosis, scleroderma, systemic sclerosis, arthral fibrosis, Peyronie's syndrome, Dupeytren's contracture, diabetic neuropathy, adhesive capsulitis, alcoholic liver disease, steatohepatitis, viral Hepatitis, biliary tract disease, primary hemochromatosis, drug-related cirrhosis, latent cirrhosis, Wilson's disease, and, alpha 1-antitrypsin deficiency, interstitial lung disease (ILD), human fibrotic lung disease, hepatic fibrosis, macular degeneration, retinoretinopathy, vitreoretinopathy, myocardial fibrosis, Graves' eye disease, drug-induced ergot poisoning, cardiovascular disease, atherosclerosis / recurrent stenosis, hypertrophic scarring, primary or idiopathic myelofibrosis, and inflammatory bowel disease (including but not limited to collagenous colitis). In some aspects, diseases associated with fibrosis originate from one or more of lung disease, lung cancer, drug therapy, chemotherapy, or radiation therapy.In some aspects, diseases associated with fibrosis originate from one or more of aging, heart attack, stroke, myocardial damage, or left ventricular dysfunction. In some aspects, diseases associated with fibrosis may be selected from renal fibrosis, glomerulonephritis, chronic kidney disease, chronic renal failure, and nephritis associated with systemic lupus erythematosus, cancer, physical disability, toxins, metabolic diseases, immune diseases, or diabetic nephropathy. In some aspects, diseases associated with fibrosis originate from one or more of 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, diseases associated with fibrosis may be selected from pulmonary fibrosis, interstitial lung disease, human fibrotic lung disease, idiopathic pulmonary fibrosis, hepatic fibrosis, cardiac fibrosis, cardiomyopathy, macular degeneration, retinoretinopathy, vitreoretinopathy, Graves' eye disease, drug-induced ergot poisoning, cardiovascular disease, atherosclerosis / recurrent stenosis, keloids and hypertrophic scars, primary or idiopathic myelofibrosis, inflammatory bowel disease, collagenous colitis, ocular scarring, and cataract fibrosis. In some aspects, diseases associated with fibrosis may be selected from NASH, hepatic fibrosis, and cirrhosis. In some aspects, diseases associated with fibrosis may be NASH. In some aspects, diseases associated with fibrosis may be selected from diabetic nephropathy, chronic kidney disease, and renal fibrosis. In some aspects, diseases associated with fibrosis may be selected from metabolic heart failure and cardiac fibrosis. In some aspects, diseases associated with fibrosis may be pulmonary fibrosis.

[0265] In some aspects, the present disclosure provides a method for reducing liver fat fraction in a subject who requires a reduction in liver fat fraction, comprising administering an effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide described herein to the subject, wherein the subject is optionally at risk of developing NASH or diagnosed with NASH.

[0266] In some aspects, the present disclosure provides a method for reducing liver stiffness and / or reducing body fat percentage, reducing body weight, reducing liver-to-body-weight ratio, reducing liver lipid content, reducing liver fibrosis area, reducing fasting blood glucose levels, reducing fasting triglycerides, reducing LDL cholesterol, reducing ApoB, reducing ApoC, and / or increasing HDL cholesterol in a subject who requires reduction in liver stiffness, reduction in body fat percentage, reduction in body weight, reduction in liver-to-body-weight ratio, reduction in liver lipid content, reduction in liver fibrosis area, reduction in fasting blood glucose levels, reduction in fasting triglycerides, reduction in LDL cholesterol, reduction in ApoB, reduction in ApoC, and / or increase in HDL cholesterol in said subject, wherein optionally the subject is diagnosed as being at risk of developing NASH or having NASH.

[0267] In some aspects, the present disclosure provides a method for increasing adiponectin levels in a subject who requires an increase in adiponectin levels, comprising administering an effective amount of a pharmaceutical formulation containing FGF-21 polypeptide to the subject, wherein optionally the subject is at risk of developing NASH or has been diagnosed with NASH.

[0268] In some aspects, the present disclosure provides a method for treating one or more symptoms associated with NASH in a subject who requires treatment for one or more symptoms associated with NASH, comprising administering an effective amount of a pharmaceutical formulation containing FGF-21 polypeptide to the subject.

[0269] Additionally, a method for treating or preventing NASH in a subject requiring treatment or prevention of NASH is provided herein, comprising administering to the subject a therapeutically effective amount of a pharmaceutical formulation disclosed herein comprising a modified FGF-21 polypeptide (PEG-FGF-21) comprising sequence identification number: 1, wherein the pAF residues thereof are connected to a poly(ethylene glycol) moiety having a molecular weight of about 30 kDa.

[0270] In some aspects, the subject may exhibit NASH CRN fibrosis stages 1–3, which is determined randomly by liver biopsy. In some aspects, prior to treatment, the subject may exhibit a fatty liver index of at least about 60. In some aspects, prior to treatment, the subject may exhibit a liver fat fraction percentage of at least 10%, which is determined randomly by magnetic resonance imaging.

[0271] In some aspects, the present disclosure provides a method for treating type 1 diabetes or type 2 diabetes in a subject who requires treatment for type 1 diabetes or type 2 diabetes, comprising administering a therapeutically effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide described herein to the subject who requires treatment for said diabetes. In some aspects, the present disclosure provides a method for treating obesity in a subject who requires treatment for said obesity, comprising administering a therapeutically effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide described herein to the subject who requires treatment for said obesity. In some aspects, the present disclosure provides a method for controlling at least one of glucose and lipid homeostasis, glucose intake, GLUT 1 expression, and / or serum concentrations of glucose, triglycerides, insulin, or glucagon in a subject who requires control of at least one of glucose and lipid homeostasis, glucose intake, GLUT 1 expression, and / or serum concentrations of glucose, triglycerides, insulin, or glucagon, comprising administering an effective amount of a pharmaceutical formulation comprising the FGF-21 polypeptide described herein to the subject.

[0272] In some aspects, the present disclosure provides a method for increasing insulin sensitivity, increasing adiponectin levels, decreasing blood glucose levels, decreasing glucagon levels, decreasing triglyceride levels, decreasing fructosamine levels, decreasing low-density cholesterol levels, or decreasing C-reactive protein levels in a subject who requires increased insulin sensitivity, increased levels of adiponectin, decreased levels of blood glucose, decreased levels of glucagon, decreased levels of triglycerides, decreased levels of fructosamine, decreased levels of low-density cholesterol, or decreased levels of C-reactive protein, by administering an effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide described herein to a subject who requires increased insulin sensitivity, increased levels of adiponectin, decreased levels of blood glucose, decreased levels of glucagon, decreased levels of triglycerides, decreased levels of fructosamine, decreased levels of low-density cholesterol, or decreased levels of C-reactive protein.

[0273] In some aspects, the present disclosure provides a method for treating a condition or disorder selected from obesity, diabetes mellitus, pancreatitis, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, impaired glucose tolerance, inappropriate glucose clearance, high blood glucose, and Prader-Willi syndrome in a subject who requires treatment for said condition or disorder selected from

[0274] In some aspects, the present disclosure provides a method for treating an insulin-related condition or disorder selected from type A insulin resistance, type C insulin resistance (AKA HAIR-AN syndrome), Labson-Mendenhall syndrome, Donohue syndrome or lepreconism, androgen excess, hirsutism, or acanthosis nigricans in a subject who requires treatment for the insulin-related condition or disorder selected from said subject, comprising administering an effective amount of a pharmaceutical formulation containing the FGF-21 polypeptide described herein to said subject who requires treatment for the insulin-related condition or disorder selected from type A insulin resistance, type C insulin resistance (AKA HAIR-AN syndrome), Labson-Mendenhall syndrome, Donohue syndrome or lepreconism, androgen excess, hirsutism, or acanthosis nigricans.

[0275] In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered by injection. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered by subcutaneous injection, IV injection, intraperitoneal injection, or intramuscular injection.

[0276] In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once per day or less than about once per day. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about twice per week or less than about twice per week. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once per week or less than about twice per week. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once every two weeks or less than about twice per week. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once every three weeks or less than about twice per week. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once per month or less than about once per month. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of once every four weeks. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once per day. In some aspects, pharmaceutical formulations containing the FGF-21 polypeptide disclosed herein may be administered at a frequency of about once per week.

[0277] In some aspects, pharmaceutical formulations comprising the FGF-21 polypeptide disclosed herein may be administered in amounts 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 FGF-21 polypeptide per dose.

[0278] For example, a pharmaceutical formulation containing the FGF-21 polypeptide disclosed herein may be administered to a subject at a concentration of FGF-21 polypeptide of about 0.1 to 100 mg / kg body weight of the recipient subject. In some aspects, a pharmaceutical formulation containing the FGF-21 polypeptide disclosed herein may be administered to a subject at a concentration of FGF-21 polypeptide of about 0.5 to 5 mg / kg body weight of the recipient subject. In another aspect, a pharmaceutical formulation containing the FGF-21 polypeptide disclosed herein may be administered to a recipient subject at a frequency of once per day to once every two weeks, e.g., about once or twice per week, once every two days, once every three days, once every four days, once every five days, or once every six days.

[0279] The pharmaceutical formulation of the present disclosure may be administered by any conventional route suitable for a protein or peptide, including but not limited to parenteral, e.g., subcutaneous or intravenous injection or any other form of injection or infusion.

[0280] IV. Modified FGF-21 polypeptide

[0281] The modified FGF-21 polypeptide of the present disclosure comprises an FGF-21 polypeptide comprising one or more amino acid substitutions, additions, or deletions. For example, the modified FGF-21 polypeptide of the present disclosure comprises modifications such as one or more amino acid substitutions (e.g., to naturally occurring or non-naturally occurring amino acids), deletions (terminal or internal deletions), or the attachment of heterogeneous moiety (by insertion / insertion into the amino acid sequence or by attachment of side chains, C-terminal, N-terminal, or internal). In some specific aspects, the modified FGF-21 polypeptide comprises a modified version of SEQ ID NO: 3 (wild-type FGF-21) or SEQ ID NO: 1 (wild-type sequence modified by substitution of glutamine 109 with para-acetyl-L-phenylalanine). In some specific aspects, the modified FGF-21 polypeptide is a derivative of sequence identification number: 1, for example, through the attachment of a heterogeneous moiety (e.g., PEG). In some specific aspects, the modified FGF-21 is PEG-FGF-21 (sequence identification number: 2).

[0282] Table 1: Sequence of FGF-21 polypeptide

[0283]

[0284] The term modified FGF-21 polypeptide also includes polymorphs (e.g., naturally occurring FGF-21 sequence variants), e.g., the P-form or L-form of FGF-21.

[0285] Substitutions at a wide variety of amino acid positions in naturally occurring FGF-21 have been described. Substitutions that modify solubility or stability, increase agonist activity, increase in vivo or in vitro half-life, increase protease resistance, convert the polypeptide into an antagonist, reduce immunogenicity or toxicity, facilitate purification or manufacturing, or provide any combination thereof are also included in the term modified FGF-21 polypeptide.

[0286] In some cases, non-naturally coded amino acid substitution(s) may be combined with other additions, substitutions, or deletions within the modified FGF-21 polypeptide to achieve different biological characteristics of the modified FGF-21 polypeptide for another FGF-21 polypeptide (e.g., the wild-type FGF-21 polypeptide of SEQ ID NO: 3, the modified FGF-21 polypeptide of SEQ ID NO: 1, the PEG-FGF-21 polypeptide of SEQ ID NO: 2, the same FGF-21 polypeptide without additions, substitutions, or deletions, or another unmodified or modified FGF-21 polypeptide).

[0287] In some cases, other additions, substitutions, or deletions may increase the stability of the modified FGF-21 polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the modified FGF-21 polypeptide for its receptor. In some cases, other additions, substitutions, or deletions may increase the pharmaceutical stability of the modified FGF-21 polypeptide. In some cases, other additions, substitutions, or deletions may increase the solubility of the modified FGF-21 polypeptide.

[0288] In some aspects, in addition to another site for the incorporation of non-natural amino acids that provides increased polypeptide solubility after expression in recombinant host cells, a site for substitution with naturally coded or non-natural amino acids is selected.

[0289] In some aspects, the modified FGF-21 polypeptide includes other additions, substitutions, or deletions that modulate the affinity for the FGF-21 polypeptide receptor, binding protein, or associated ligand, modulate post-binding signaling to the FGF-21 receptor, modulate the circulating half-life, modulate release or bioavailability, facilitate purification, or improve or change a specific route of administration.

[0290] In some aspects, the modified FGF-21 polypeptide comprises additions, substitutions, or deletions that increase the affinity of the modified FGF-21 for its receptor. Similarly, the modified FGF-21 polypeptide may comprise chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to FLAG or poly-His), or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.), or linked molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, size reduction of the modified FGF-21, or other characteristics of the polypeptide.

[0291] Multiple polymorphs of FGF-21 have been identified. Leucine or proline is described at the same location in U.S. Patent Publication No. 20010012628 and U.S. Patent No. 6,716,626. N-terminal leader or signal sequences with a single amino acid (leucine) differing from each other appear in U.S. Patent No. 6,716,626 and U.S. Patent Publication No. 20040259780. FGF-21 polypeptide variants or mutations are described in U.S. Patent No. 6,716,626; U.S. Patent Publications No. 2005 / 0176631, 2005 / 0037457, 2004 / 0185494, 2004 / 0259780, 2002 / 0164713, and 2001 / 0012628; WO 01 / 36640; WO 03 / 011213; WO 03 / 059270; WO 04 / 110472; WO 05 / 061712; WO 05 / 072769; WO 05 / 091944; WO 05 / 113606; WO 06 / 028595; WO 06 / 028714; WO 06 / 050247; WO 06 / 065582; WO 06 / 078463; WO 01 / 018172; WO 09 / 149171; WO 10 / 042747; WO 12 / 066075; WO 11 / 154349; WO 13 / 052311; WO 13 / 188181 (the full text thereof is incorporated herein by reference) includes, but is not limited to.

[0292] The term modified FGF-21 polypeptide also includes biologically active fragments, biologically active variants, and stereoisomers of naturally occurring FGF-21, as well as agonist, mimic, and antagonist variants of naturally occurring FGF-21 and their polypeptide fusions. Fusions containing additional amino acids at the amino terminus, carboxyl terminus, or both are also included in the term modified FGF-21 polypeptide.

[0293] Exemplary fusions include, for example, methionyl FGF-21 with methionine linked to the N-terminus of FGF-21 derived from recombinant expression of a mature form of FGF-21 without a leader or signal peptide or part thereof (e.g., FGF-21 with methionine linked to the N-terminus of FGF-21 derived from recombinant expression in E. coli), fusions for purification (including but not limited to poly-histidine or affinity epitopes), fusions with serum albumin-binding peptides such as PKE adnectin and fusions with serum proteins such as serum albumin, and fusion proteins including but not limited to FGF-21 and serum albumin, Fc domain, immunoglobulin constant region, unstructured polypeptide, e.g. XTEN, etc. (heteromoty). Any such fragment can be prepared from a protein by standard biochemical methods or by the expression of a polynucleotide encoding the fragment.

[0294] The term modified FGF-21 polypeptide comprises polypeptides conjugated to a polymer such as PEG, which may optionally include one or more additional derivatizations of cysteine, lysine, or other residues. Additionally, the modified FGF-21 polypeptide may comprise a linker or a polymer (wherein the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid according to the present disclosure), or may be conjugated to a naturally encoded amino acid using techniques known in the art, such as coupling to lysine or cysteine.

[0295] The term modified FGF-21 polypeptide also includes glycosylated modified FGF-21, e.g., polypeptides glycosylated at any amino acid position (but not limited thereto), N-linked or O-linked glycosylated forms of the polypeptide. Modifiers containing a single nucleotide change are also considered as biologically active modifications of the FGF-21 polypeptide. Additionally, splice modifications are also included.

[0296] The term modified FGF-21 polypeptide also includes any one or more unmodified or modified FGF-21 polypeptides or any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or any type of other biologically active molecule expressed as a fusion protein linked by chemical means, as well as FGF-21 polypeptide heteromers, homomers, heteropolymers, or homopolymers of any other polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or any other type of biologically active molecule, and polypeptide analogs containing, for example, specific deletions or other modifications also retain biological activity.

[0297] The term modified FGF-21 polypeptide also includes FGF-21 polypeptide comprising one or more amino acid substitutions, insertions, or deletions. For example, the modified FGF-21 polypeptide of the present disclosure may consist of one or more modifications to natural amino acids, optionally with one or more non-natural amino acid modifications. The term modified FGF-21 polypeptide also includes exemplary substitutions, insertions, or deletions at a wide variety of amino acid positions within the FGF-21 polypeptide that modify pharmaceutical stability, modify one or more of the biological activities of the FGF-21 polypeptide, increase agonist activity (though not limited thereto), increase the solubility of the polypeptide, decrease protease sensitivity, decrease deamidation, convert the polypeptide into an antagonist, decrease immunogenicity or toxicity, or facilitate purification or manufacturing.

[0298] In some aspects, the modified FGF-21 polypeptide further comprises additional insertions, substitutions, or deletions that modulate the biological activity of the modified FGF-21 polypeptide. For example, additions, substitutions, or deletions may modulate one or more properties or activities of the modified FGF-21. For example, additions, substitutions, or deletions may modulate the affinity for the FGF-21 polypeptide receptor, modulate the cyclic half-life, modulate the therapeutic half-life, modulate the stability of the polypeptide, modulate protease cleavage, modulate the dose, modulate the release or bioavailability, facilitate purification, reduce deamidation, improve the use-life, or improve or change a specific route of administration. Similarly, the modified FGF-21 polypeptide may include a protease cleavage sequence, a reactive group, an antibody-binding domain (including but not limited to FLAG or poly-His) or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.) or a linked molecule (including but not limited to biotin) that improves detection (including but not limited to GFP), purification, or other characteristics of the polypeptide.

[0299] The term modified FGF-21 polypeptide also includes homodimers, heterodimers, homopolymers, and heteropolymers formed through linked, or fused partners, such as the Fc domain, which include but are not limited to those directly linked to the same or different non-naturally coded amino acid side chains, to the natural-coded amino acid side chains, or indirectly linked through a linker. Exemplary linkers include, but are not limited to, small organic compounds, water-soluble polymers of various lengths, such as poly(ethylene glycol) or polydextran, or polypeptides of various lengths.

[0300] In some aspects, the non-naturally coded amino acids described herein may comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. In some aspects, the non-naturally coded amino acids comprise a carbonyl moiety and are linked to a linker, polymer, biologically active molecule, or half-life extension moiety comprising an aminooxy, hydrazine, hydrazide, or semicarbazide moiety. In some aspects, the non-naturally coded amino acids comprise an aminooxy, hydrazine, hydrazide, or semicarbazide moiety linked to a linker, polymer, biologically active molecule, or half-life extension moiety via an amide linkage.

[0301] In some aspects, the non-naturally coded amino acid comprises an alkyne moiety that is connected to a linker, polymer, biologically active molecule, or half-life extension moiety via an azide moiety. In some aspects, the non-naturally coded amino acid comprises an azide moiety that is connected to a linker, polymer, biologically active molecule, or half-life extension moiety containing an alkyne moiety. In some cases, one non-naturally coded amino acid comprises an azide or alkyne moiety that is connected to a linker, polymer, biologically active molecule, or half-life extension moiety via an amide linkage.

[0302] In some aspects, a non-naturally coded amino acid is connected to a linker, polymer, biologically active molecule, or half-life extension moiety via an oxime linkage. In some aspects, a non-naturally coded amino acid is connected to a linker, polymer, biologically active molecule, or half-life extension moiety via an oxime linkage, wherein the oxime linkage has a structure derived from the reaction of a carbonyl group and an aminooxy group. In some aspects, a non-naturally coded amino acid is connected to a linker, polymer, biologically active molecule, or half-life extension moiety via an oxime linkage, wherein the oxime linkage has a structure derived from the reaction of a carbonyl group contained within the non-naturally coded amino acid and an aminooxy group contained within the linker, polymer, biologically active molecule, or half-life extension moiety.

[0303] In some aspects, the modified FGF-21 polypeptide described herein has at least one biological activity of the wild-type human FGF-21 polypeptide having the amino acid sequence of SEQ ID No. 3.

[0304] In some aspects, the modified FGF-21 polypeptide comprises at least one non-naturally encoded amino acid linked to a water-soluble polymer containing a poly(ethylene glycol) moiety.In some aspects, one or more non-naturally coded amino acids are incorporated into one or more of the following positions within the modified FGF-21: prior to 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, 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, 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., at the carboxyl terminus of the protein) (amino acid position corresponds to sequence identification number: 3).

[0305] In some aspects, the FGF-21 polypeptide in the pharmaceutical formulations disclosed herein is, for example, U.S. Patent No. 9,273,106; 9,458,214; 9,744,213; 8,541,369; 8,188,040; 9,120,871; 9,895,417; 9,266,935; 6,716,626; 4,904,584; or 8,722,622; U.S. Application Publication No. 2013 / 0231277; 2009 / 0305986; 2011 / 0195895; 2010 / 0216715; 2001 / 0012628; 2004 / 0259780; It is a modified FGF-21 polypeptide described in 2005 / 0176631; 20070293430; or 2012 / 0035099; or PCT publication number WO2016048999; WO2010065439; WO2017093465; WO2003011213; WO2003059270; WO2005091944; WO2008121563; WO199967291, WO199903887, WO200026354; or WO2017220706 (all of which the full text is incorporated herein by reference).

[0306] V. Half-life extension motility

[0307] The FGF-21 polypeptide of the present disclosure may comprise a heterogeneous moiety, which in some aspects is a half-life extension moiety. In some aspects, the half-life extension moiety is a polypeptide moiety, and in some other aspects, the half-life extension moiety is a non-polypeptide moiety. In some aspects, the FGF-21 polypeptide comprises a single half-life extension moiety. However, in other aspects, the FGF-21 polypeptide disclosed herein may comprise more than one half-life extension moiety.

[0308] Half-life extension moiety includes non-protein and protein half-life extension moiety. Non-protein half-life extension moiety is, for example, water-soluble polymer, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), lipids, branched or unbranched acyl groups, branched or unbranched C8-C 30 Acyl group, branched or unbranched alkyl group, and branched or unbranched C8-C 30 It includes alkyl groups. Protein half-life extension moiety includes, for example, serum albumin, transferrin, adnectin (e.g., albumin-binding or pharmacokinetic extension (PKE) adnectin), Fc domains, and unstructured polypeptides, such as XTEN and PAS (e.g., shape-disordered polypeptide sequences composed of amino acids Pro, Ala, and / or Ser), as well as fragments of any of these.

[0309] Examination of the crystal structure of FGF-21 or FGF family member(s) and their interaction with FGF receptors can indicate whether specific amino acid residues possess side chains that are fully or partially accessible to the solvent. Side chains of non-naturally encoded amino acids at these positions may be directed away from the protein surface into the solvent and, thus, may be linked, for example, to a water-soluble polymer.

[0310] (a) Immunoglobulin invariant region or part thereof

[0311] In some aspects, the FGF-21 polypeptide is linked, for example, fused or conjugated to the immunoglobulin constant region or a part thereof. The immunoglobulin constant region consists of domains represented by CH (constant heavy chain) domains (CH1, CH2, etc.). Depending on the isotype (i.e., IgG, IgM, IgA, IgD, or IgE), the constant region may consist of three or four CH domains. Some isotypes (e.g., IgG) constant regions also contain hinge regions. [Janeway] et al. 2001, Immunobiology , Garland Publishing, NY, NY].

[0312] The immunoglobulin constant region or a portion thereof for the generation of the fusion protein of the present disclosure may be obtained from many different sources. In some aspects, the immunoglobulin constant region or a portion thereof is derived from human immunoglobulin. However, it is understood that the immunoglobulin constant region or a portion thereof may be derived from immunoglobulin of other mammalian species, including, for example, rodent species (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, macaque). Additionally, the immunoglobulin constant region or a portion thereof may be derived from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In one aspect, human isotype IgG1 is used.

[0313] Various immunoglobulin constant region gene sequences (e.g., human constant region gene sequences) are available in the form of publicly accessible deposits. The constant region domain sequence may be selected to have a specific effector function (or lack a specific effector function) or to have a specific modification to reduce immunogenicity. Many sequences of antibodies and antibody-coding genes have been disclosed, and suitable Ig constant region sequences (e.g., hinge, CH2, and / or CH3 sequences, or parts thereof) may be derived from these sequences using techniques recognized in the relevant art. Subsequently, the genetic material obtained using any of the above methods may be modified or synthesized to obtain the polypeptide of the present disclosure. Additionally, it will be recognized that the scope of the present disclosure includes alleles, modifications, and mutations of the constant region DNA sequence.

[0314] The sequence of an immunoglobulin constant region or a part thereof can be cloned, for example, using a polymerase chain reaction and primers selected to amplify the domain of interest. To clone the sequence of an immunoglobulin constant region or a part thereof from an antibody, mRNA can be isolated from hybridomas, spleens, or lymphocytes, reverse transcribed into DNA, and the antibody gene amplified by PCR. PCR amplification methods are described in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188; also, for example, in the literature ["PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, CA (1990)]; [Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. As described in [217:270]. PCR can be initiated by common constant region primers or more specific primers based on the disclosed heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding antibody light and heavy chains. In this case, the library can be screened by common primers or larger homologous probes, such as mouse constant region probes. Many primer sets suitable for the amplification of antibody genes are known in the relevant art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250).The cloning of the antibody sequence is further described in U.S. Patent No. 5,658,570 (Newman et al., filed January 25, 1995).

[0315] The immunoglobulin constant region used herein may include all domains and hinge regions or parts thereof. In one aspect, the immunoglobulin constant region or parts thereof includes a CH2 domain, a CH3 domain, and a hinge region, i.e., an Fc region or an FcRn binding partner.

[0316] As used herein, the term “Fc region” is defined as a part of a polypeptide formed by the dimeric association of the Fc domains of each of the two heavy chains of a native immunoglobulin corresponding to the Fc region of the native immunoglobulin. The native Fc region forms a homodimer with another Fc region.

[0317] In one aspect, the "Fc region" refers to a portion of a single immunoglobulin heavy chain that starts at the hinge region immediately upstream of the papain cleavage site (i.e., residue 216' in IgG, with the first residue of the heavy chain constant region being 114) and terminates at the C-terminus of the antibody. Thus, the complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain.

[0318] The Fc region of the immunoglobulin constant region may include CH2, CH3, and CH4 domains, as well as a hinge region, depending on the immunoglobulin isotype. Fusion proteins containing the Fc region of the immunoglobulin confer several desirable properties to the fusion protein, including increased stability, increased serum half-life (see reference [Capon et al., 1989, Nature 337:525]), as well as binding to Fc receptors, such as neogenic Fc receptors (FcRn) (U.S. Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03 / 077834; US2003-0235536A1).

[0319] In some aspects, the "Fc region" comprises an amino acid sequence of or derived from the Fc domain. In certain aspects, the Fc region comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain (approximately amino acids 216–230 of the antibody Fc region according to EU numbering), a CH2 domain (approximately amino acids 231–340 of the antibody Fc region according to EU numbering), a CH3 domain (around amino acids 341–438 of the antibody Fc region according to EU numbering), a CH4 domain, or a variant, part, or fragment thereof. In other aspects, the Fc region comprises a complete Fc domain (i.e., the hinge domain, the CH2 domain, and the CH3 domain). In some aspects, the Fc region comprises, is essentially composed of, or is composed of a hinge domain (or part thereof) fused to a CH3 domain (or part thereof), a hinge domain (or part thereof) fused to a CH2 domain (or part thereof), a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof), or a CH2 domain (or part thereof) fused to both a hinge domain (or part thereof) and a CH3 domain (or part thereof). Also in other aspects, the Fc region does not have at least a part of a CH2 domain (e.g., all or part of a CH2 domain). In certain aspects, the Fc region comprises or is composed of amino acids corresponding to EU numbers 221 through 447.

[0320] The Fc domain, denoted herein by F, F1, or F2, can be obtained from many different sources. In one aspect, the Fc region of the polypeptide is derived from human immunoglobulin. However, it is understood that the Fc region may be derived from immunoglobulin of other mammalian species, including, for example, rodent species (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, macaque). Additionally, the polypeptide of the Fc domain or a part thereof may be derived from any class of immunoglobulin, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In another aspect, human isotype IgG1 is used.

[0321] In a specific aspect, the Fc variant provides at least one effector function modification conferred by the Fc region comprising the wild-type Fc domain (e.g., improvement or reduction in the ability of the Fc region to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complementary protein (e.g., C1q), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC). In another aspect, the Fc variant provides a engineered cysteine ​​residue.

[0322] The Fc region of the present disclosure may use art-recognized Fc variations known to impart (e.g., enhancement or reduction) to the effector function and / or FcR combination. Specifically, the binding molecules of the present disclosure are, for example, International PCT disclosures WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2; US Patent Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; Or it may include a change (e.g., substitution) at one or more of the amino acid positions disclosed in U.S. Patents 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; 7,404,956, and 7,317,091.In one aspect, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the relevant art) may occur at one or more of the disclosed amino acid positions. In another aspect, a different change at one or more of the disclosed amino acid positions (e.g., a different substitution of one or more amino acid positions disclosed in the relevant art) may occur.

[0323] The Fc region of IgG can be modified according to widely recognized procedures, such as site-directed mutagenesis, to obtain a modified IgG or Fc fragment or part thereof that binds to the Fc receptor. Such modifications include modifications within the contact site that preserve or even enhance binding to the Fc receptor, as well as modifications located far from the Fc receptor contact site.For example, the following single amino acid residues in human IgG1 Fc (Fcγ1) can be substituted without a significant loss of Fc binding affinity to the Fc receptor: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where, for example, P238A represents wild-type proline substituted by alanine at position number 238. For example, a specific aspect removes a highly conserved N-glycosylation site and incorporates the N297A mutation. In addition to alanine, other amino acids may substitute for the wild-type amino acid at the positions specified above.Mutations may be introduced into Fc to form more than 100 Fc regions different from the native Fc. Additionally, combinations of two, three, or more of these individual mutations may be introduced together to form hundreds of additional Fc regions. Furthermore, one of the Fc regions of the construct of the present disclosure may be mutated while other Fc regions of the construct may not be mutated at all, or both may be mutated by different mutations.

[0324] Certain of the above-mentioned mutations can provide novel functionality to the Fc region. For example, one aspect removes a highly conserved N-glycosylation site and incorporates N297A. The effect of this mutation is to improve the circulating half-life of the Fc region by reducing immunogenicity, and also to make the Fc region unable to bind to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA without loss of affinity (Routledge et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632; Shields et al. 1995, J. Biol. Chem. 276:6591). As an example of the addition of novel functionality resulting from the mutations described above, the affinity for the Fc receptor may increase in some cases beyond that of the wild type. This increased affinity may reflect an increased "on" rate, a decreased "off" rate, or both an increased "on" rate and a decreased "off" rate. Examples of mutations believed to confer increased affinity for the Fc receptor include, but are not limited to, T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276:6591).

[0325] Additionally, at least three human Fc gamma receptors appear to recognize binding sites on IgG within the basal hinge region, typically at amino acids 234–237. Therefore, another example of novel functionality and potential reduced immunogenicity may arise from mutations in this region, for instance, by replacing the amino acid sequence “ELLG” at amino acids 233–236 of human IgG1 with the corresponding sequence “PVA” from IgG2 (a one-amino acid deletion). It has been shown that FcγRII, FcγRII, and FcγRIII, which mediate various effector functions, would not bind to IgG1 upon the introduction of these mutations. See [Ward and Ghetie 1995, Therapeutic Immunology 2:77] and [Armour et al. 1999, Eur. J. Immunol. 29:2613].

[0326] In certain aspects, the immunoglobulin constant region or a part thereof is semi-glycosylated. For example, a fusion protein comprising two Fc regions or FcRn binding partners may contain a first, glycosylated, Fc region (e.g., a glycosylated CH2 region) and a second, non-glycosylated, Fc region (e.g., a non-glycosylated CH2 region). In one aspect, a linker may be interposed between the glycosylated Fc region and the non-glycosylated Fc region. In another aspect, the Fc region or FcRn binding partner is fully glycosylated, that is, all Fc regions are glycosylated. In another aspect, the Fc region may be non-glycosylated, that is, the Fc moiety is not glycosylated.

[0327] In certain aspects, the fusion protein of the present disclosure comprises an amino acid substitution (e.g., an Fc variant) for an immunoglobulin constant region or a part thereof that alters the antigen-independent effector function of the Ig constant region, in particular the cyclic half-life of the protein.

[0328] These proteins exhibit increased or decreased binding to Fc receptors compared to proteins without these substitutions, and thus have increased or decreased serum half-lives, respectively. Fc variants with improved affinity for Fc receptors are expected to have longer serum half-lives, and these molecules have useful applications in the treatment of mammals where a long half-life of the administered polypeptide is desired, e.g., in the treatment of chronic diseases or disorders (see, e.g., U.S. Patent Nos. 7,348,004, 7,404,956, and 7,862,820). On the other hand, Fc variants with reduced Fc receptor binding affinity are expected to have shorter half-lives, and these molecules are also useful, e.g., for administration to mammals where a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging, or in situations where toxic side effects occur when the starting polypeptide is present in circulation for an extended period. Fc variants with reduced Fc receptor binding affinity are also less likely to cross the placenta and are therefore also useful in the treatment of diseases or disorders in pregnant women.

[0329] Additionally, other applications where reduced Fc receptor binding affinity may be desired include applications where localization in the brain, kidney, and / or liver is desired. In one exemplary aspect, the fusion protein of the present disclosure exhibits reduced transport from the vascular system across the epithelium of the renal glomerulus. In another aspect, the fusion protein of the present disclosure exhibits reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one aspect, a protein having modified Fc receptor binding comprises at least one Fc region (e.g., one or two Fc regions) having one or more amino acid substitutions within the "Fc receptor binding loop" of the Ig constant region. The Fc receptor binding loop consists of amino acid residues 280–299 (according to EU numbering) of the wild-type, full-length Fc region.

[0330] In another aspect, the Ig constant region of the present disclosure having a modified Fc receptor binding affinity or a part thereof comprises at least one Fc region having one or more amino acid substitutions at amino acid positions corresponding to any one of the following EU positions: 256, 277-281, 283-288, 303-309, 313, 338, 342, 376, 381, 384, 385, 387, 434 (e.g., N434A or N434K), and 438. Exemplary amino acid substitutions having a modified Fc receptor binding activity are disclosed in International PCT Publication No. WO05 / 047327.

[0331] (b) albumin or a fragment thereof, or a variant thereof

[0332] In some aspects, the FGF-21 polypeptide is linked to albumin or its functional fragments, for example, fused or conjugated.

[0333] Human serum albumin (HSA, or HA), a protein of 609 amino acids in its full-length form, is responsible for a significant portion of the osmotic pressure of serum and also functions as a carrier for endogenous and exogenous ligands. As used herein, the term “albumin” includes full-length albumin or its functional fragments, modifications, derivatives, or analogs.

[0334] In some aspects, the half-life extension moiety linked to the FGF-21 polypeptide is albumin or a fragment or variant thereof, which extends (or can extend) the half-life of the FGF-21 polypeptide. Examples of albumin or a fragment or variant thereof are disclosed in U.S. Patent Publication No. 2008 / 0194481 A1, 2008 / 0004206 A1, 2008 / 0161243 A1, 2008 / 0261877 A1, or 2008 / 0153751 A1 or PCT Application Publication No. 2008 / 033413 A2, 2009 / 058322 A1, or 2007 / 021494 A2.

[0335] (c) Albumin-binding moiety

[0336] In certain aspects, the half-life extension moiety linked to the FGF-21 polypeptide, e.g., fused or conjugated, is an albumin-binding moiety comprising an albumin-binding peptide, a bacterial albumin-binding domain, an albumin-binding antibody fragment, or any combination thereof. For example, the albumin-binding protein may be a bacterial albumin-binding protein, an antibody, or an antibody fragment comprising a domain antibody (see U.S. Patent No. 6,696,245). The albumin-binding protein may be, for example, a bacterial albumin-binding domain, such as one of Streptococcus protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218, 73-83). Other examples of albumin-binding peptides that may be used as conjugation partners are, for example, U.S. Patent Application 2003 / 0069395 or the literature [Dennis et al. (2002) J. Biol. Chem. These have the common sequence Cys-Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Cys as described in [277, 35035-35043], where Xaa 1 is Asp, Asn, Ser, Thr, or Trp; Xaa 2 is Asn, Gln, His, Ile, Leu, or Lys; Xaa 3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa 4 is Asp, Gly, Leu, Phe, Ser, or Thr.

[0337] (d) PAS sequence

[0338] In another aspect, the half-life extension moiety linked to the FGF-21 polypeptide, for example, fused or conjugated, is a PAS sequence. As used herein, "PAS sequence" means an amino acid sequence comprising, or comprising, mainly alanine and serine residues, or primarily alanine, serine, and proline residues, which forms a random coil shape under physiological conditions. Thus, a PAS sequence is a building block, amino acid polymer, or sequence cassette comprising, or essentially composed of, alanine, serine, and proline, which can be used as part of a heterogeneous moiety in a fusion protein. Furthermore, a person skilled in the art recognizes that an amino acid polymer may also form a random coil shape when residues other than alanine, serine, and proline are added to the PAS sequence as incidental components. As used herein, the term "incidental component" refers to the fact that amino acids other than alanine, serine, and proline may be added to the PAS sequence to some extent, for example, up to about 12%, i.e., about 12 of 100 amino acids of the PAS sequence; up to about 10%, i.e., about 10 of 100 amino acids of the PAS sequence; up to about 9%, i.e., about 9 of 100 amino acids; up to about 8%, i.e., about 8 of 100 amino acids; about 6%, i.e., about 6 of 100 amino acids; about 5%, i.e., about 5 of 100 amino acids; about 4%, i.e., about 4 of 100 amino acids; about 3%, i.e., about 3 of 100 amino acids; about 2%, i.e., about 2 of 100 amino acids; and about 1%, i.e., about 1 of 100 amino acids. Amino acids different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val.

[0339] Under physiological conditions, PAS sequence elongation forms a random coil shape, which can thereby mediate increased in vivo and / or in vitro stability for the FGF-21 polypeptide. Since the random coil domain does not adopt a stable structure or function in itself, the biological activity mediated by FGF-21 is essentially preserved. In another respect, the PAS sequence forming the random coil domain is biologically inactive, particularly regarding proteolysis in plasma, immunogenicity, isoelectric / electrostatic behavior, and binding or internalization to cell surface receptors, yet remains biodegradable, which provides a clear advantage over synthetic polymers such as PEG.

[0340] Exemplary PAS sequences are provided in, for example, U.S. Patent Publication No. 2010 / 0292130 A1 and PCT Application Publication No. WO 2008 / 155134 A1 (the full text of both of which is incorporated herein by reference).

[0341] (e) HAP sequence

[0342] In certain aspects, a half-life extension moiety linked to the FGF-21 polypeptide, e.g., fused or conjugated, is a glycine-rich homo-amino-acid polymer (HAP). The HAP sequence may contain repeat sequences of glycine, which have a length of at least 50 amino acids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160 amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500 amino acids.

[0343] In one aspect, an HAP sequence can extend the half-life of a moiety fused or linked to the HAP sequence. Non-limiting examples of HAP sequences include, but are not limited to, (Gly)n, (Gly4Ser)n, or S(Gly4Ser)n (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In one aspect, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In another aspect, n is 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

[0344] (f) transferrin or its fragment

[0345] In certain aspects, the half-life extension moiety linked to the FGF-21 polypeptide, e.g., fused or conjugated, is transferrin or a fragment thereof. Any transferrin may be used to form the fusion protein of the present disclosure. For example, wild-type human Tf (Tf) is a 679-amino acid protein of approximately 75 kDa (glycosylation not considered) having two major domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to be derived from a gene duplication. See GenBank accession numbers NM001063, XM002793, M12530, XM039845, XM039847 and S95936 (www.ncbi.nlm.nih.gov / ). Transferrin comprises two domains, an N domain and a C domain. The N domain includes two subdomains, the N1 domain and the N2 domain, and the C domain includes two subdomains, the C1 domain and the C2 domain.

[0346] In one aspect, the transferrin portion of the fusion protein comprises a transferrin splice variant. In one example, the transferrin splice variant may be a splice variant of human transferrin, GenBank accession AAA61140. In another aspect, the transferrin portion of the fusion protein comprises one or more domains of the transferrin sequence, e.g., an N domain, a C domain, an N1 domain, an N2 domain, a C1 domain, a C2 domain, or any combination thereof.

[0347] (g) Polymer, e.g., polyethylene glycol (PEG)

[0348] In another aspect, a half-life extending moiety linked to the FGF-21 polypeptide, e.g., fused or conjugated, is a soluble polymer known in the art, including but not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, or polyvinyl alcohol. Chemically modified derivatives of the FGF-21 polypeptide of this disclosure may provide additional benefits such as increased solubility, stability and cycle time of the polypeptide, or reduced immunogenicity (see U.S. Patent No. 4,179,337). The soluble polymer may be attached to any position within the FGF-21 polypeptide or at any position of its N- or C-terminus. The soluble polymer may be attached to a random position within the FGF-21 polypeptide sequence or to a predetermined position within the FGF-21 polypeptide and may comprise one, two, three, or more attached soluble polymer moietys. In some aspects, the polymer is attached to the side chain of a naturally occurring amino acid. In other aspects, the polymer is attached to the side chain of a non-naturally encoded amino acid, for example, a phenylalanine derivative, such as para-acetyl-L-phenylalanine.

[0349] Soluble polymers can have any molecular weight and can be branched or unbranched. In the case of PEG, in one aspect, the molecular weight is about 1 kDa to about 100 kDa for ease of handling and manufacturing. In some aspects, the molecular weight of a soluble polymer, e.g., PEG, is about 30 kDa. Depending on the desired profile (e.g., desired duration of sustained release, effect on biological activity if present, ease of handling, degree or absence of antigenicity and other known effects of polyethylene glycol on proteins or analogs), other sizes may be used. For example, PEG is approximately 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, It can have an average molecular weight of 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 Da.

[0350] In some respects, PEG can have a branched structure. Branched PEG is described, for example, in U.S. Patent 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)].

[0351] The number of PEG moieties attached to the FGF-21 polypeptide may also vary. For example, the PEGylated protein of the present disclosure may, on average, be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more PEG molecules. Similarly, the average degree of substitution may be within the range of 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 PEG moieties per protein molecule. A method for determining the degree of substitution is disclosed, for example, in the literature [Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992)].

[0352] In another aspect, the FGF-21 polypeptide used in the formulations of the present disclosure is conjugated to one or more polymers. The polymers are water-soluble and may be covalently or non-covalently attached to the FGF-21 polypeptide or other moiety conjugated to the FGF-21 polypeptide. Non-limiting examples of the polymers may be poly(alkylene oxide), poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine).

[0353] (h) Hydroxyethyl starch (HES)

[0354] In certain aspects, half-life-extending moiety linked to the FGF-21 polypeptide, for example, fused or conjugated, is hydroxyethyl starch (HES) or its derivatives. HES is a derivative of naturally occurring amylopectin and is degraded by alpha-amylase in the body. HES is a substituted derivative of amylopectin, a carbohydrate polymer present in corn starch at concentrations of 95 wt% or less. HES exhibits advantageous biological properties and is used as a blood volume replacement agent, as well as in hemodilation therapy in clinics (references [Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987)]; and [Weidler et al., Arzneim.-Forschung / Drug Res., 41, 494-498 (1991)]).

[0355] Amylopectin contains a glucose moiety in which alpha-1,4-glycosidic bonds are present within the main chain and alpha-1,6-glycosidic bonds appear at branching sites. The physicochemical properties of this molecule are primarily determined by the type of glycosidic bonds. Due to the nicked alpha-1,4-glycosidic bonds, a helical structure with approximately six glucose monomers per turn is generated. The physicochemical as well as biochemical properties of the polymer can be modified through substitution. The introduction of hydroxyethyl groups can be achieved through alkaline hydroxyethylation. By adapting the reaction conditions, the different reactivity of each hydroxyl group within the unsubstituted glucose monomer to hydroxyethylation can be utilized. Due to this fact, a person skilled in the art can influence the substitution pattern to a limited extent.

[0356] HES is primarily characterized by molecular weight distribution and degree of substitution. The degree of substitution, denoted by DS, relates to molar substitution and is known to those skilled in the art. See the literature cited above [Sommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), in particular p. 273].

[0357] In one aspect, the HES has an average molecular weight (weight average) of 1 to 300 kD, 2 to 200 kD, 3 to 100 kD, or 4 to 70 kD. The hydroxyethyl starch may further exhibit a molar degree of substitution of 0.1 to 3, preferably 0.1 to 2, more preferably 0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6 substitutions in the range of 2 to 20 for the hydroxyethyl group. Non-limiting examples of HES having an average molecular weight of about 130 kD are HES having a degree of substitution of 0.2 to 0.8, e.g. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferably 0.4 to 0.7, e.g. 0.4, 0.5, 0.6, or 0.7. Specifically, an HES with an average molecular weight of about 130 kD is Voluven® from Fresenius. Voluven® is an artificial colloid used for volume replacement, for example, in therapeutic indications for the treatment and prevention of hypovolemia. The characteristics of Voluven® are an average molecular weight of 130,000+ / -20,000 D, a molar substitution of 0.4, and a C2:C6 ratio of about 9:1. In another aspect, the range of average molecular weight of hydroxyethyl starch is, for example, 4 to 70 kD or 10 to 70 kD or 12 to 70 kD or 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to 50 kD or 12 to 50 kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or 12 to 18 kD or 4 to 12 kD or 10 to 12 kD or 4 to 10 kD.Also, in another aspect, the average molecular weight of the hydroxyethyl starch used is in the range of greater than 4 kD and less than 70 kD, e.g., about 10 kD, or 9 to 10 kD, or 10 to 11 kD, or 9 to 11 kD, or about 12 kD, or 11 to 12 kD, or 12 to 13 kD, or 1 to 13 kD, or about 18 kD, or 17 to 18 kD, or 18 to 19 kD, or 17 to 19 kD, or about 30 kD, or 29 to 30, or 30 to 31 kD, or about 50 kD, or 49 to 50 kD, or 50 to 51 kD, or 49 to 51 kD.

[0358] In certain aspects, the half-life extension moiety may be a mixture of HES having different average molecular weights and / or different degrees of substitution and / or different ratios of C2:C6 substitution. Accordingly, mixtures of HES having different average molecular weights and different degrees of substitution and different ratios of C2:C6 substitution, or different average molecular weights and different degrees of substitution and the same or approximately the same ratio of C2:C6 substitution, or different average molecular weights and the same or approximately the same degree of substitution and the same ratio of C2:C6 substitution, or the same or approximately the same average molecular weights and different degrees of substitution and the same ratio of C2:C6 substitution, or different average molecular weights and the same or approximately the same degree of substitution and the same or approximately the same ratio of C2:C6 substitution, or the same or approximately the same average molecular weights and different degrees of substitution and the same or approximately the same ratio of C2:C6 substitution, or the same or approximately the same average molecular weights and the same or approximately the same degree of substitution and the different ratios of C2:C6 substitution, or the approximately the same average molecular weights and approximately the same degree of substitution and the approximately the same ratio of C2:C6 substitution.

[0359] (i) Polysialic acid (PSA)

[0360] In certain aspects, half-life-extending moiety linked to the FGF-21 polypeptide, for example, fused or conjugated, is polysialic acid (PSA) or its derivatives. PSA is a naturally occurring unbranched polymer of sialic acid produced in certain cells in mammals and also by certain bacterial strains (Roth J., et al. (1993), Polysialic Acid: From Microbes to Man, eds Roth J., Rutishauser U., Troy FA (Birkhaeuser Verlag, Basel, Switzerland), pp 335-348). These can be produced by restricted acid hydrolysis, digestion with neuraminidase, or fractionation of the natural, bacterial-derived forms of the polymer, with varying degrees of polymerization from n=about 80 or more sialic acid residues up to n=2.

[0361] The composition of different polysial acids also varies, such that alpha-2,8-linked polysial acids, including the encapsulated polysaccharides of E. coli strain K1 and Group B meningococcus, also appear in homopolymer forms, i.e., in the embryonic form of the neuronal adhesion molecule (N-CAM). Heterogeneous forms also exist (e.g., alternating alpha-2,8 alpha-2,9 polysial acids of Group C polysaccharides of E. coli strain K92 and N. meningitidis). Sial acids may also appear in alternating copolymers with monomers other than sialic acid, such as Group Y or Group W135 of N. meningitidis. Polysial acids have important biological functions, including evasion of immune and complementary systems by pathogenic bacteria and regulation of glial adhesion of immature neurons during fetal development (where the polymer has an anti-adhesion function) (Cho and Troy, PNAS, USA, 91 (1994) 11427-11431), but there are no known receptors for polysial acids in mammals. The alpha-2,8-linked polysial acid of E. coli strain K1 is also known as 'colomic acid,' which is used to exemplify the present disclosure (in various lengths).

[0362] Various methods for attaching or conjugating polysialic acid to polypeptides are described (see, e.g., U.S. Patent No. 5,846,951; WO-A-0187922, and US 2007 / 0191597 A1).

[0363] (j) XTEN sequence

[0364] In some aspects, the half-life extension moiety linked to the FGF-21 polypeptide, for example, fused or conjugated, is the XTEN sequence. As used herein, "XTEN sequence" refers to an extended-length polypeptide having a non-naturally occurring, substantially non-repetitive sequence composed mainly of small hydrophilic amino acids, having no or minimal secondary or tertiary structure under physiological conditions. As a fusion protein partner, XTEN may be provided as a carrier linked to the FGF-21 polypeptide to provide certain desirable pharmacokinetic, physicochemical, and pharmaceutical properties upon the formation of the fusion protein. These desirable properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics.

[0365] In some aspects, the XTEN sequence of the present disclosure is a peptide or polypeptide having about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or more than 2000 amino acid residues. In certain aspects, XTEN is a peptide or polypeptide having more than about 20 to about 3000 amino acid residues, more than 30 to about 2500 residues, more than 40 to about 2000 residues, more than 50 to about 1500 residues, more than 60 to about 1000 residues, more than 70 to about 900 residues, more than 80 to about 800 residues, more than 90 to about 700 residues, more than 100 to about 600 residues, more than 110 to about 500 residues, or more than 120 to about 400 residues.

[0366] The XTEN sequence of the present disclosure may comprise one or more sequence motifs of 9 to 14 amino acid residues or an amino acid sequence that is at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence motif, wherein the motif comprises, is essentially composed of, or is composed of 4 to 6 types of amino acids selected from the group consisting of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P). See US 2010-0239554 A1.

[0367] In some aspects, the XTEN sequence comprises a number of units of non-overlapping sequence motifs of the AD motif family, or the AE motif family, or the AF motif family, or the AG motif family, or the AM motif family, or the AQ motif family, or the BC family, or the BD family, wherein the XTEN generated herein represents a range of homologues. In other aspects, the XTEN comprises a number of units of motif sequences from two or more of the motif families. These sequences may be selected to achieve desired physical / chemical characteristics, including properties such as net charge, hydrophilicity, absence of secondary structure, or absence of repeatability, provided by the amino acid composition of the motifs, which are described in more detail below. In other aspects, the motifs incorporated into the XTEN may be selected and assembled using the methods described herein to achieve XTENs of about 36 to about 3000 amino acid residues.

[0368] In a further aspect, the XTEN sequence used in this disclosure affects the physical or chemical properties, e.g., pharmacokinetics, of the fusion protein of this disclosure. The XTEN sequence used in this disclosure may exhibit one or more of the following advantageous properties: shape flexibility, enhanced water solubility, a high degree of protease resistance, low immunogenicity, low binding to mammalian receptors, or increased hydrodynamic (or Stokes) radius.

[0369] In some aspects, an XTEN sequence linked to an FGF-21 polypeptide can enhance pharmacokinetic properties (e.g., a longer termination half-life or an increased area under the curve (AUC)), and thus the FGF-21 polypeptide remains in vivo for an increased duration compared to the corresponding FGF-21 polypeptide without XTEN. In additional aspects, an XTEN sequence linked to an FGF-21 polypeptide disclosed herein can enhance pharmacokinetic properties (e.g., a longer termination half-life or an increased area under the curve (AUC)), and thus the FGF-21 polypeptide remains in vivo for an increased duration compared to the corresponding FGF-21 polypeptide without XTEN.

[0370] The physical and chemical properties of proteins containing the XTEN sequence can be determined using various methods and assays. These methods include, but are not limited to, analytical centrifugation, EPR, HPLC-ion exchange, HPLC-size exclusion, HPLC-reverse phase, light scattering, capillary electrophoresis, circular dichroism, differential scanning calorimetry, fluorescence HPLC-ion exchange, HPLC-size exclusion, IR, NMR, Raman spectroscopy, refractography, and UV / visible spectroscopy. Additional methods are disclosed in the literature [Amau et al., Prot Expr and Purif 48, 1-13 (2006)].

[0371] Additional examples of XTEN sequences that may be used according to the present disclosure are disclosed in US Patent Publication No. 2010 / 0239554 A1, 2010 / 0323956 A1, 2011 / 0046060 A1, 2011 / 0046061 A1, 2011 / 0077199 A1, or 2011 / 0172146 A1, or International Patent Publication No. WO 2010091122 A1, WO 2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, or WO 2011028344 A2, all of which are incorporated herein by reference in their entirety.

[0372] (k) Immunoglobulin-binding peptide (or polypeptide)

[0373] In certain aspects, a half-life extension moiety linked to the FGF-21 polypeptide, for example, fused or conjugated, is an immunoglobulin-binding peptide. The immunoglobulin-binding peptide can bind to the Fc region and improve the half-life of the fusion protein described herein.

[0374] In some aspects, the immunoglobulin-binding peptide useful in the present disclosure is a peptide or polypeptide having about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or more than 500 amino acid residues.

[0375] In some aspects, the immunoglobulin-binding peptide useful in the present disclosure comprises a 13-mer IgG-Fc domain-binding peptide (IgGBP). Reference [DeLano WL, et al. (2000) Science 287:1279-1283]. In other aspects, the immunoglobulin-binding peptide useful in the present disclosure comprises the peptide disclosed in U.S. Patent Publication No. 20170334954, U.S. Patent Publication No. 20170210777, or PCT Publication No. WO / 2017 / 069158.

[0376] VI. Manufactured Articles and Kits

[0377] The present disclosure also provides an FGF-21 pharmaceutical formulation comprising (i) the FGF-21 polypeptide disclosed herein (e.g., modified FGF-21, e.g., PEG-FGF-21), and (ii) a manufacturing article and kit comprising instructions for use. The manufacturing article may include a container. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from various materials, such as glass, plastic, or metal. The container holds an FGF-21 pharmaceutical formulation, for example, a liquid formulation.

[0378] A label on or combined with the container may indicate instructions for reconstitution and / or use. For example, the label may indicate that the FGF-21 pharmaceutical preparation should be diluted to a protein concentration as described above. The label may additionally indicate that a subcutaneous preparation is useful or intended for subcutaneous administration.

[0379] The container holding the formulation may be, for example, a multi-use vial that enables repeated administration (e.g., 2 to 6 doses, or more) of the subcutaneous formulation. Alternatively, the container may be, for example, a pre-filled syringe containing the subcutaneous formulation.

[0380] The manufactured article or kit may further comprise, for example, a second container containing a solvent. The manufactured article may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts having instructions for use.

[0381] It should be acknowledged that the detailed description section, rather than the summary and summary section, is intended to be used to interpret the claims. The summary and summary may describe one or more, but not all, exemplary aspects of the present disclosure considered by the inventor(s), and accordingly, are not intended to limit the present disclosure and the appended claims in any way.

[0382] The present disclosure is described above with the assistance of functional building blocks representing the execution of specified functions and their relationships. The boundaries of these functional building blocks are defined arbitrarily herein for convenience of explanation. Alternative boundaries may be defined as long as the specified functions and their relationships are properly performed.

[0383] The foregoing description of specific aspects will reveal the general nature of the present disclosure in great detail, and other aspects can be easily modified and / or adapted for various applications without departing from the general concept of the present disclosure and without excessive experimentation by applying knowledge within the art in the relevant technical field. Accordingly, such adaptations and modifications are intended to be within the scope of the meaning and equivalents of the disclosed aspects based on the teachings and guidelines provided herein. It should be understood that phrases or terms used herein are for illustrative purposes only and not limiting, and accordingly, terms or phrases in this specification should be interpreted by a person skilled in the art in light of the teachings and guidelines.

[0384] The scope and range of the present disclosure shall not be limited by any of the exemplary aspects described above, but shall be defined only by the following claims and their equivalents.

[0385] Examples

[0386] Example 1

[0387] Development of PEG-FGF-21 formulations

[0388] For initial studies, PEG-FGF-21 was formulated at 7.5 mg / mL in 20 mM Tris, 250 mM sucrose, and pH 8.3. Due to the high deamidation rate observed in this formulation, the drug product had to be frozen and stored at -20°C. Therefore, additional work was performed to develop a ready-to-use (RTU) formulation that would allow the drug product to be stored at 2-8°C.

[0389] PEG-FGF-21 (SEQ ID: 2) was prepared by attaching a linear 30-kDa PEG moiety to FGF-21 (SEQ ID: 1) modified in a site-specific manner. To achieve site-specific PEGylation, the activated PEG was reacted with the non-natural amino acid, para-acetylphenylalanine (pAF), at position 109 of the FGF-21 molecule by a method known in the art to form a chemically stable oxime-linked PEG-protein conjugate.

[0390] Lower formulation pH was shown to increase the rate of molecular aggregation, while higher formulation pH was shown to increase the rate of deamidation. At a target protein concentration of 10 mg / mL, pH 7.0 was found to be the best balance between the rates of aggregation and deamidation. Increasing the sucrose concentration was shown to further stabilize the molecule against aggregation.

[0391] The formulation selected for further study was 10 mg / mL PEG-FGF-21 in 20 mM L-histidine, 600 mM sucrose, and pH 7.0. Considering a weekly dose of 20 mg, higher concentrations of the drug product in the pre-filled syringe were developed. To enable higher concentrations of the drug product, further optimization of the formulation was essential.

[0392] The initial development of higher concentration formulations was carried out in 3 mL Type I glass vials. Upon stirring of 15 mg / mL and 22 mg / mL PEG-FGF-21 in 20 mM L-histidine, 600 mM sucrose, and pH 7.0, bubble capture was observed and the solution became increasingly turbid (Fig. 5, left image). In an attempt to alleviate the problem, polysorbate 80 was used. Polysorbate 80 was able to prevent bubble capture (Fig. 5, right image). Therefore, polysorbate 80 was added to the high concentration formulations of PEG-FGF-21 at a concentration of 0.05% (w / v).

[0393] Oxidation is an important quality property of PEG-FGF-21, and the oxidation of methionine-169 near the C-terminus destroys receptor binding and consequently the activity of the molecule. Metal-catalyzed oxidation is one of the mechanisms by which proteins can be oxidized. Trace concentrations of metal may be present in protein preparations due to residues from cell culture, contact with stainless steel containers, impurities in excipients, and the formation process of pre-fillable syringes.

[0394] To test whether PEG-FGF-21 is susceptible to metal-catalyzed oxidation, samples with and without a metal cocktail (250 ppm Fe, 10 ppm Cu, 15 ppm Cr, 15 ppm Ni, 10 ppm Mo) were incubated at 25°C for 1 month and at 40°C for 2 weeks, respectively, in the presence or absence of 0.05 mM pentetate (diethylenetriaminepentaacetic acid, DTPA), and the oxidation levels of methionine 1 and methionine 169 were determined by trypsin peptide mapping (Figs. 2a and 2b).

[0395] In the absence of pentetate (DTPA), significant oxidation of both methionine residues was observed even in samples without added metal, and the oxidation levels were much higher in samples spiked with a metal cocktail. In samples containing 50 μM pentetate, no increase in oxidation levels was observed for both methionines. This indicates that PEG-FGF-21 is susceptible to metal-catalyzed oxidation and that trace levels of metal are already present in the formulation. Pentetate effectively chelated not only the metal present in the formulation but also additional metals spiked into the formulation, preventing metal-catalyzed oxidation of methionines 1 and 169. Therefore, 50 μM pentetate was added to a high-concentration formulation of PEG-FGF-21.

[0396] To optimize the pH for higher concentration formulations, the inventors reviewed aggregation data of PEG-FGF-21 collected at pH 6.3 to 8.5, which showed that the aggregation rate increased rapidly at pH 6.8 or lower (Figs. 3a and 3b). Accordingly, the inventors increased the target pH for high concentration formulations to pH 7.0 to pH 7.1.

[0397] To compare the new high-concentration formulation with the previous formulation, 20 mg / mL of PEG-FGF-21 (before pH optimization) in 20 mM L-histidine, 600 mM sucrose, 0.05 mM pentetate, 0.05% (w / v) polysorbate 80, and pH 7.0 was placed for stability and compared with 22.5 mg / mL of PEG-FGF-21 in 20 mM L-histidine, 600 mM sucrose, and pH 7.0.

[0398] As shown in Figures 4a and 4b and Figures 1a and 1b, the addition of polysorbate 80 and pentetic acid to the PEG-FGF-21 formulation provided lower aggregation rates at all temperatures and less deamidation at 40°C.

[0399] Test method

[0400] aggregation Aggregation was tested by size exclusion high-performance liquid chromatography on a commercially available system (e.g., Agilent Technologies 1100 Series HPLC system with PDA detector or Waters Alliance e2695 Series HPLC with PDA detector) equipped with a commercially available analytical column (Tosoh TSK Gel G3000SWXL, 7.8 x 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 into each experiment. The amount of aggregate was determined using instrument software by dividing the area of ​​the high molecular weight peak by the total area of ​​all observed peaks.

[0401] Deamidation: Deamidation was tested by anion exchange high-performance liquid chromatography on a commercially available system (e.g., Agilent Technologies 1100 Series HPLC system with PDA detector or Waters Alliance e2695 Series HPLC with PDA detector) equipped with a commercially available analytical column (Agilent Bio Wax, non-porous, 5 μm column, 4.6 x 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 modifications were separated using a linear gradient from 2% B to 67% B over 20 minutes at a flow rate of 1.0 ml / min. The samples were diluted to a protein concentration of 1 mg / mL with mobile phase A, and 0.075 mg (0.075 mL) was injected into each experiment. Using instrument software, the amount of deamidation was determined by dividing the area of ​​the acidic modified peak by the total area of ​​all observed peaks.

[0402] oxidationOxidation was tested by trypsin peptide mapping. After digesting protein samples with trypsin, the resulting peptides were separated by reverse-phase ultra-high performance liquid chromatography on a commercially available system (e.g., Waters Acquity UPLC) equipped with a commercially available analytical column (Waters Acquity C18 BEH Peptide RP UPLC column, 2.1 x 150 mm, 1.7-μm particle size, 130-Å pore size 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. Trypsin peptides were separated using a complex gradient from 10% B to 40% B over 27 minutes at a temperature of 60°C at a flow rate of 0.3 ml / min. The amount of oxidation was determined by using instrument software to divide the area of ​​the oxidized peptide peak by the total area of ​​the oxidized and corresponding non-oxidized peaks.

[0403] Particle formation and bubble formation: Visual observation.

[0404] Concentration of PEG-FGF-21 : The concentration of PEG-FGF-21 in the pharmaceutical formulation disclosed herein was measured by slope spectroscopy at 280 nm using an absorption coefficient of 0.87 (mL / (mg*cm)).

[0405] Determination of pH pH is the standard method (USP <791> It was decided according to ).

[0406] Concentration of excipients : The concentration of individual excipients (e.g., DTPA, PS80, histidine, sucrose) in the pharmaceutical formulations disclosed herein was determined / calculated as the amount (weight, mole, etc.) of the individual excipient added to the pharmaceutical formulation during the manufacturing process per unit of the final volume of the finished pharmaceutical formulation. Alternatively, the concentration of the excipient may be based on the actual amount of the individual excipient in the pharmaceutical formulation.

Claims

Claim 1 (i) PEGylated fibroblast growth factor 21 (FGF-21) polypeptide (PEG-FGF-21) consisting of the sequence described in SEQ ID No. 2, at a concentration of 20 mg / mL when measured by slope spectroscopy at 280 nm with an extinction coefficient of 0.87 mL / (mg*cm); (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) a pharmaceutical formulation comprising diethylenetriaminepentaacetic acid (DTPA) at a concentration of 50 μM, wherein the pH is 7.1 and DTPA functions as a structural stabilizer, wherein the formulation: (a) a lower deamidation rate of PEGylated FGF-21 polypeptide compared to a reference formulation not containing DTPA when stored at 40°C for about 1 month; (b) a lower aggregation rate of high molecular weight (HMW) PEGylated FGF-21 polypeptide compared to a reference formulation not containing DTPA when stored at 40°C for about 1 month; or (c) a pharmaceutical formulation exhibiting both of (a) and (b). Claim 2 In paragraph 1, a pharmaceutical preparation in which the preparation is formulated for subcutaneous administration. Claim 3 A pharmaceutical preparation for use in the treatment or prevention of diseases or conditions associated with fibrosis and / or diabetes mellitus, as described in claim 1. Claim 4 A pharmaceutical preparation for use in the treatment or prevention of diabetes or non-alcoholic fatty liver disease (NASH) according to claim 1. Claim 5 A method for preparing a formulation according to claim 1, comprising mixing PEGylated fibroblast growth factor 21 (FGF-21) polypeptide with DTPA. Claim 6 (i) a pharmaceutical preparation according to paragraph 1, and (ii) a kit including instructions for use. Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete Claim 98 delete Claim 99 delete Claim 100 delete Claim 101 delete Claim 102 delete Claim 103 delete Claim 104 delete Claim 105 delete Claim 106 delete Claim 107 delete Claim 108 delete Claim 109 delete Claim 110 delete Claim 111 delete Claim 112 delete Claim 113 delete Claim 114 delete Claim 115 delete Claim 116 delete Claim 117 delete Claim 118 delete Claim 119 delete Claim 120 delete Claim 121 delete Claim 122 delete Claim 123 delete Claim 124 delete Claim 125 delete Claim 126 delete Claim 127 delete Claim 128 delete Claim 129 delete Claim 130 delete

Images