Liquid formulations comprising PEGylated conjugates of mutant FGF-21 peptides

Abstract

The disclosure relates to a liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate and a pre-filled syringe or autosyringe comprising a liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate.

Description

[0001] Case information

[0002] This application is a divisional application of the invention patent application with application number 202280027650.8 filed on March 10, 2022 and invention name “Liquid preparation comprising a mutant FGF-21 peptide PEGylated conjugate”.

[0003] Related Applications

[0004] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 159,717, filed on March 11, 2021, and U.S. Provisional Application No. 63 / 167,148, filed on March 29, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

[0005] Sequence Listing

[0006] This application includes a sequence listing filed herein, which includes the file 180234-011502PCT_ST25.txt having the following size 43,360 bytes, which was created on March 3, 2022, the contents of which are hereby incorporated herein by reference. Technical Field

[0007] The disclosure relates to a pharmaceutical liquid formulation comprising a recombinant fibroblast growth factor-21 (FGF-21) peptide PEGylated conjugate and a prefilled syringe or autoinjector comprising a liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate. Background Art

[0008] FGF-21 is an endocrine hormone naturally found as a monomeric non-glycosylated protein. Together with FGF-19 and FGF-23, FGF-21 belongs to the endocrine action subfamily, and the remaining 18 mammalian FGF ligands are grouped into five paracrine action subfamilies. Compared with the paracrine action FGF, the endocrine action FGF only shows low affinity to heparin sulfate, and therefore can enter the blood circulation. Therefore, endocrine FGF can regulate metabolic processes, such as bile acid homeostasis, liver glucose and protein metabolism (FGF-19), glucose and lipid metabolism (FGF-21) and vitamin D and phosphate homeostasis (FGF-23). Summary of the invention

[0009] Aspects of the disclosure relate to a prefilled syringe or autoinjector comprising a liquid pharmaceutical composition comprising: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant fibroblast growth factor-21 peptide conjugate comprising a mutant FGF-21 peptide comprising an amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between a threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG. PEG; (b) 150mM to 500mM arginine; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) 5 to 25mM buffer, pH 7-8; and (e) a pharmaceutically acceptable carrier. In some embodiments, the liquid pharmaceutical composition in the prefilled syringe or automatic injector comprises about 28mg / mL of a mutant FGF-21 peptide conjugate, about 260mM arginine HCl, about 20mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition in the prefilled syringe or auto-injector comprises about 20 mg / mL of mutant FGF-21 peptide conjugate, about 150 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.5. In some embodiments, the liquid pharmaceutical composition in the prefilled syringe or auto-injector comprises about 36 mg / mL of mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition in the prefilled syringe or auto-injector comprises about 44 mg / mL of mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition in a prefilled syringe or autoinjector comprises about 44 mg / mL of a mutant FGF-21 peptide conjugate, about 230 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1.

[0010] Aspects of the disclosure relate to a liquid pharmaceutical composition comprising: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant fibroblast growth factor-21 peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between a threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. PEG; (b) 150 mM to 500 mM arginine; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) 5 to 25 mM buffer, pH 7-8; and (e) a pharmaceutically acceptable carrier.

[0011] Other aspects of the disclosure relate to a liquid pharmaceutical composition comprising: (a) 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant fibroblast growth factor-21 peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between a threonine at amino acid position 173 of SEQ ID NO:2 and a first site of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second site of the glycosyl moiety and the 20 kDa PEG. PEG; (b) 150 mM to 500 mM arginine, 50 mM to 250 mM alanine, 50 mM to 250 mM proline, 50 mM to 250 mM glycine, 50 mM to 250 mM MgCl2, 1 to 5% (v / v) glycerol, 1 to 5% (v / v) PEG 400, or a combination thereof; (c) 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); (d) a buffer at pH 7-8; and (e) a pharmaceutically acceptable carrier.

[0012] In some embodiments, the liquid preparation further comprises a surfactant. In some embodiments, the surfactant comprises hexadecyltrimethylammonium bromide, sodium gluconate, or a combination thereof. In some embodiments, the liquid preparation comprises 0.05% to 0.1% (w / v) hexadecyltrimethylammonium bromide, 0.05% to 0.1% (w / v) sodium gluconate, or a combination thereof.

[0013] In some embodiments, the buffer is Tris or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the pH of the liquid formulation is 7.0 to 7.5.

[0014] In some embodiments, the liquid pharmaceutical composition comprises 20 to 44 mg / ml of the mutant FGF-21 peptide conjugate.

[0015] In some embodiments, the liquid pharmaceutical composition comprises 150 mM to 275 mM arginine. In some embodiments, the arginine in the liquid pharmaceutical composition comprises arginine HCl, arginine sulfate, or a combination thereof. In some embodiments, the weight ratio of the mutant FGF-21 peptide conjugate to arginine is 0.6 to 0.9. In some embodiments, the molar ratio of the mutant FGF-21 peptide conjugate to arginine is about 0.006 to about 0.009.

[0016] In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL of a mutant FGF-21 peptide conjugate, about 260 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1.

[0017] In some embodiments, the liquid pharmaceutical composition comprises about 20 mg / mL of a mutant FGF-21 peptide conjugate, about 150 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.5.

[0018] In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL of a mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1.

[0019] In some embodiments, the liquid pharmaceutical composition comprises about 44 mg / mL of a mutant FGF-21 peptide conjugate, about 200 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1.

[0020] In some embodiments, the liquid pharmaceutical composition comprises about 44 mg / mL of a mutant FGF-21 peptide conjugate, about 230 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.1.

[0021] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0022] In some embodiments, the liquid pharmaceutical composition is stable for up to 12 months at a temperature in the range of 2° C. to 8° C. In some embodiments, the liquid pharmaceutical composition is stable at room temperature for at least 3 months.

[0023] In some embodiments, a container comprising a liquid pharmaceutical composition is provided. In some embodiments, the container is a prefilled syringe, vial, or autoinjector. In some embodiments, a kit is provided, which includes a container and a label or instructions for administering and using the liquid pharmaceutical composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Among the benefits and improvements disclosed, other objects and advantages of the present invention will become apparent from the following description in conjunction with the accompanying drawings. Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely illustrative of the present invention and that the present invention can be implemented in various forms. In addition, each example given in conjunction with various embodiments of the present invention is intended to be illustrative rather than restrictive.

[0025] Figure 1 The structure of a specific embodiment of the invention, namely a mutant FGF-21 peptide conjugate comprising the exemplary structure of FGF-21 (Thr) -GalNAc-Sia-Gly-PEG is shown. n is selected to give the desired PEG molecular weight. For 20 kDa PEG, n is within the range selected from 450 to 460.

[0026] Figure 2 A non-limiting exemplary cell expansion, production, and harvesting process flow diagram is shown according to some embodiments.

[0027] Figure 3 A non-limiting exemplary refolding and mutant FGF-21 peptide purification process flow diagram according to some embodiments is shown.

[0028] Figure 4 A non-limiting exemplary GlycoPEGylation and final purification process flow diagram according to some embodiments is shown.

[0029] Figure 5 Shown are the average Z-average diameter (nm) results for buffer types according to some embodiments.

[0030] Figure 6 Shown are average polydispersity index (PDI) results for buffers according to some embodiments.

[0031] Figure 7 An overlay of three sample traces obtained for 20 mM Tris, 150 mM Arginine HCl, pH 8.0 is shown, indicating acceptable polydispersity according to some embodiments.

[0032] Figure 8 Shown is the percent monomeric PEGylated DS used for solubility screening according to some embodiments.

[0033] Fig. 9 Shown is the percentage dimer PEGylated DS used for solubility screening according to some embodiments.

[0034] Fig.10 Shown is the percent total aggregate PEGylated DS used for solubility screening according to some embodiments.

[0035] Fig.11 An overlay of three sample traces obtained for 20 mM Tris, 150 mM Arginine HCl, pH 8.0 is shown, with the lines indicating the Tm of each triplicate reading according to some embodiments.

[0036] Fig.12 Shown is a 3D surface map of a differential scanning fluorimetry (DSF) TO according to some embodiments.

[0037] Fig.13 Dynamic light scattering (DLS) 2W / 5°C overall PDI is shown according to some embodiments.

[0038] Fig.14A Shown is the low molecular weight (LMW) at 2W / 5°C by size exclusion chromatography (SEC) according to some embodiments.

[0039] Fig. 14B Shown is the LMW at 2W / 25°C by SEC according to some embodiments.

[0040] Fig.15 Shown is a 3D surface map -5°C of main peak percentage (MP) imaging capillary isoelectric focusing (icIEF) according to some embodiments.

[0041] Fig.16 Shown is a 3D surface of SEC % MP 2W / 25°C according to some embodiments.

[0042] Fig.17 Shown is SDS-PAGE reducing gel-12M stability (TO) according to some embodiments.

[0043] Fig.18Shown is SDS-PAGE non-reducing gel-12M stability (T0) according to some embodiments.

[0044] Fig.19 Shown is an SDS-PAGE reducing gel-12M stability (T12M) according to some embodiments.

[0045] Fig. 20 Shown is SDS-PAGE non-reducing gel-12M stability (T12M) according to some embodiments.

[0046] Fig.21 Shown is the effect of Arginine HCl on dimerization according to some embodiments.

[0047] Figure 22A-22C Shown is a response surface plot from a DOE study for optimizing PEG-FGF21 formulations according to some embodiments. DETAILED DESCRIPTION

[0048] Among those benefits and improvements disclosed, other objects and advantages of the disclosure will become apparent from the following description in conjunction with the accompanying drawings. Detailed embodiments of the disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely illustrative of the disclosure that can be embodied in various forms. In addition, each example is provided for various embodiments of the disclosure that are intended to be illustrative rather than limiting.

[0049] Throughout the specification and claims, the following terms adopt the meanings clearly associated herein, unless the context clearly provides otherwise. The phrases "in one embodiment," "in an embodiment," and "in some embodiments" used herein do not necessarily refer to the same embodiment, although they may refer to the same embodiment. In addition, the phrases "in another embodiment" and "in some other embodiments" used herein do not necessarily refer to different embodiments, although they may refer to different embodiments. All embodiments of the disclosure are intended to be combinable without departing from the scope or spirit of the disclosure.

[0050] As used herein, the term "based on" is not exclusive and allows for being based on other factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural references. The meaning of "in..." includes "in..." and "on...".

[0051] As used herein, terms such as "comprising," "including," and "having" do not limit the scope of a particular claim to the materials or steps listed in the claim.

[0052] As used herein, the term "consisting essentially of" limits the scope of a particular claim to the specified materials or steps and those that do not materially affect one or more basic and novel characteristics of the particular claim.

[0053] As used herein, terms such as "consisting of and "composed of" limit the scope of a particular claim to the materials and steps recited in that claim.

[0054] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0055] definition

[0056] For clarity and readability, the following definitions are provided. Any technical features mentioned for these definitions can be read in each and all embodiments of the present invention. Additional definitions and explanations can be specifically provided in the context of these embodiments. Unless otherwise defined, the meaning of all technical and scientific terms used herein is generally the same as the meaning generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the terms used herein and laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known in the art and commonly used. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and methods are generally carried out according to conventional methods in the art and various general references (such as Sambrook et al., 1989, Molecular Cloning Laboratory Guide, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which are provided in this document.

[0057] Enzyme: An enzyme is a catalytically active biomolecule that performs a biochemical reaction such as the transfer of a glycosyl moiety or a modified glycosyl moiety from a corresponding glycosyl donor to an amino acid of FGF-21 or to another glycosyl moiety attached to a peptide.

[0058] Protein: A protein generally comprises one or more peptides or polypeptides. Proteins are generally folded into a three-dimensional form, which may be necessary for the protein to exert its biological function. The sequence of a protein or peptide is generally understood as the order, i.e. the continuous sequence of its amino acids.

[0059] Recombinant protein: The term "recombinant protein" refers to a protein or variant of such a protein produced in a heterologous system, i.e., in an organism that does not naturally produce such a protein, i.e., the protein or peptide is "recombinantly produced." Typically, heterologous systems used in the art to produce recombinant proteins are bacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), or certain mammalian cell culture lines.

[0060] Expression host: Expression host refers to the organism used for recombinant protein production. Common expression hosts are bacteria such as Escherichia coli, yeasts such as Saccharomyces cerevisiae or Pichia pastoris, or mammalian cells such as human cells. RNA, mRNA: RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer composed of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers, which are connected to each other along the so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (i.e. ribose) of the first adjacent monomer and the phosphate part of the second adjacent monomer. A specific continuous sequence of monomers is called an RNA sequence.

[0061] DNA: DNA is a common abbreviation for deoxyribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are usually deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate, and deoxy-cytidine-monophosphate monomers, which themselves are composed of a sugar portion (deoxyribose), a base portion, and a phosphate portion and are polymerized by a characteristic backbone structure. The backbone structure is usually formed by a phosphodiester bond between the sugar portion of the nucleotide, i.e., the deoxyribose of the first adjacent monomer, and the phosphate portion of the second adjacent monomer. The specific order of monomers, i.e., the order of bases attached to the sugar / phosphate backbone, is called the DNA sequence. DNA can be single-stranded or double-stranded. In double-stranded form, the nucleotides of the first strand are usually hybridized with the nucleotides of the second strand, for example, by A / T base pairing and G / C base pairing.

[0062] Sequence of a nucleic acid molecule / nucleic acid sequence: The sequence of a nucleic acid molecule is generally understood to be a specific and individual order, ie a contiguous sequence of its nucleotides.

[0063] Sequence of an amino acid molecule / amino acid sequence: The sequence of a protein or peptide is generally understood to be the order, ie the consecutive sequence of its amino acids.

[0064] Sequence identity: If two or more sequences show the same length and order of nucleotides or amino acids, they are identical. The percentage of identity usually describes the degree to which two sequences are identical, i.e. it typically describes the percentage of nucleotides corresponding to the same nucleotides of a reference sequence (such as a natural or wild-type sequence) in its sequence position. In order to determine the degree of identity, it is considered that the sequences to be compared show the same length, i.e. the length of the longest sequence in the sequences to be compared. This means that a first sequence consisting of 8 nucleotides / amino acids is 80% identical to a second sequence consisting of 10 nucleotides / amino acids comprising the first sequence. In other words, in the context of the open text, the identity of a sequence specifically relates to the percentage of nucleotides / amino acids in sequences with the same position in two or more sequences of the same length. Gaps are generally considered to be non-identical positions, regardless of their actual positions in the alignment.

[0065] Newly introduced amino acid: "Newly introduced amino acid" means an amino acid that is newly introduced into an amino acid sequence compared to the native / wild-type amino acid sequence. The native amino acid sequence is altered, usually by mutagenesis, in order to have certain amino acid side chains at desired positions within the amino acid sequence. In the disclosure, in particular, the amino acid threonine is newly introduced into the amino acid sequence on the C-terminal side adjacent to a proline residue.

[0066] Functional group: This term is to be understood according to the general understanding of those skilled in the art and refers to a chemical moiety that is present on a molecule, particularly on a peptide or an amino acid of a peptide or a glycosyl residue attached to a peptide, and that can participate in a covalent or non-covalent bond with another chemical molecule (i.e., it allows for the attachment of, for example, a glycosyl residue or PEG).

[0067] Native amino acid sequence: This term is understood according to the general understanding of a person skilled in the art and means an amino acid sequence in the form in which it occurs in nature without any mutation or artificial amino acid modification. It is also referred to as a "wild-type sequence". "Native FGF-21" or "wild-type FGF-21" means FGF-21 having an amino acid sequence as it occurs in nature, such as the (unmutated) amino acid sequence of human FGF-21 depicted in SEQ ID NO: 1. The presence or absence of an N-terminal methionine (depending on the expression host used) does not generally alter the state of a protein that is considered to have its native or native / wild-type sequence.

[0068] Mutation: This term is to be understood according to the general understanding of a person skilled in the art. An amino acid sequence is referred to as "mutated" if it comprises at least one additional, deleted or exchanged amino acid in its amino acid sequence compared to its natural or native amino acid sequence, i.e. if the amino acid sequence comprises an amino acid mutation. A mutated protein is also referred to as a mutant. In the public text, a mutated FGF-21 peptide is in particular a peptide having an exchange of the amino acid adjacent to the proline residue on the C-terminal side of the proline residue. Thus, a consensus sequence for O-linked glycosylation is introduced into FGF-21, so that the mutant FGF-21 peptide comprises a newly introduced O-linked glycosylation side. Amino acid exchange is generally indicated as follows: S 172 T, which means that the amino acid serine at position 172 (such as in the amino acid sequence of SEQ ID NO: 1) is exchanged by the amino acid threonine.

[0069] Pharmaceutically effective amount: A pharmaceutically effective amount in the context of the disclosure is generally understood to be an amount sufficient to induce a pharmaceutical effect.

[0070] Therapy / treatment: The term "therapy" refers to "management" or "treatment" of a disease or condition, inhibiting the disease (slowing or stopping its development), alleviating the symptoms or side effects of the disease (including palliative care), and palliating the disease (causing regression of the disease).

[0071] Therapeutically effective amount: is the amount of a compound sufficient to treat a disease or condition, inhibit a disease or condition, alleviate the symptoms or side effects of a disease, and / or cause regression of a disease or condition.

[0072] Half-life: The term "half-life" as used herein in the context of administration of a mutant FGF-21 peptide and / or its conjugate is defined as the time required for the plasma concentration of the drug (i.e., mutant FGF-21 peptide and / or conjugate) to be halved in a subject.

[0073] O-linked glycosylation: "O-linked glycosylation" occurs at serine or threonine residues (Tanner et al., Biochimica et Biophysica Acta, 906:81-91 (1987); and Hounsell et al., J. Glycoplexa, 13:19-26 (1996)). In the public text, O-linked glycosylation sites (which are amino acid motifs in the amino acid sequence of a peptide that are recognized by glycosyltransferases as the attachment point of a glycosyl residue) include the amino acid motif proline-threonine (PT) that is not present in the native / wild-type amino acid sequence. In particular, a threonine residue is newly introduced near a proline and on the C-terminal side of a proline residue. The glycosyl moiety is then attached to the -OH group of the threonine residue by a glycosyltransferase.

[0074] Newly introduced O-linked glycosylation side: "Newly introduced O-linked glycosylation side" refers to an O-linked glycosylation side that is not present in native or wild-type FGF-21 prior to the introduction of a threonine near and on the C-terminal side of a proline residue as described herein.

[0075] Adjacent: Adjacent refers to an amino acid that is immediately adjacent to another amino acid in an amino acid sequence, on the N-terminal side or on the C-terminal side of the corresponding amino acid. In the disclosure, for example, a newly introduced threonine residue is adjacent to a proline residue on the C-terminal side of a proline residue.

[0076] Glycosyl moiety: The glycosyl moiety is a moiety composed of one or more identical or different glycosyl residues that connects the mutant FGF-21 peptide to polyethylene glycol (PEG), thereby forming a conjugate comprising the peptide, the glycosyl moiety and the PEG. The glycosyl moiety can be a mono-, di-, tri- or oligosaccharide moiety. The glycosyl moiety can include one or more sialic acid residues, one or more N-acetylgalactosamine (GalNAc) residues, one or more galactose (Gal) residues, etc. The glycosyl moiety can be modified, such as by PEG or methoxy-PEG (m-PEG), an alkyl derivative of PEG.

[0077] Glycoconjugation: As used herein, "glycoconjugation" refers to the enzymatically mediated conjugation of a PEG-modified glycosyl moiety to an amino acid or glycosyl residue of a (poly)peptide, such as the mutant FGF-21 of the disclosed text. A subgenus of "glycoconjugation" is "glycosyl-PEGylation", in which the modifying group of the modified glycosyl moiety is PEG or m-PEG. PEG can be linear or branched. Typically, branched PEG has a central branched core portion and multiple linear polymer chains connected to the central branched core. PEG is typically used in branched forms, which can be prepared by adding ethylene oxide to different polyols (such as glycerol, pentaerythritol, and sorbitol). The central branch portion can also be derived from a variety of amino acids, such as lysine. Branched PEG can be represented by the common form R(-PEG-OX) m, wherein R represents a core part such as glycerol or pentaerythritol, X represents an end-capping group or an end group, and m represents the number of arms. The terms "sugar-PEG" and "glycosyl-PEG" are used interchangeably, and represent a chemical moiety composed of a PEG or methoxy-PEG (mPEG or m-PEG), one or more glycosyl residues (or glycosyl moieties) and optionally a joint (such as amino acids, for example glycine) between PEG / methoxy-PEG and glycosyl moieties. The embodiment of glycosyl-PEG / sugar-PEG moieties is PEG-sialic acid (PEG-Sia). It should be noted that the terms "sugar-PEG" and "glycosyl-PEG" and "PEG-sialic acid" and "PEG-Sia" together with similar terms for sugar-PEG moieties may include or may not include a joint between PEG and one or more glycosyl moieties, i.e., "PEG-sialic acid" encompasses, for example, PEG-sialic acid and PEG-Gly sialic acid and mPEG-Gly-sialic acid.

[0078] Sequence motif: A sequence motif represents a short amino acid sequence, such as a short amino acid sequence containing only two amino acids, which is present at any possible position in a longer amino acid sequence, such as the amino acid sequence of human FGF-21. A sequence motif is represented, for example, as P 172 T, which means that the proline at position 172 is followed by a threonine residue at the C-terminus.

[0079] Sialic acid: The term "sialic acid" or "Sia" refers to any member of a family of nine-carbon carboxylated sugars. The most common member of the sialic acid family is N-acetyl-neuraminic acid (2-keto-5-acetylamino-3,5-dideoxy-D-glycero-D-galactononulopyranosyl-l-keto acid (commonly abbreviated Neu5Ac, NeuAc, or NANA). The second member of the family is N-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. The third sialic acid family member is 2-keto-3-deoxy-nonulosonic acid (nonulosonic acid). acid) (KDN) (Nadano et al. (1986) Journal of Biol. Chem. 261: 11550-11557). Also included are 9-substituted sialic acids, such as 9-O-C1-C6 acyl-Neu5Ac, such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For a review of the sialic acid family, see, for example, Varki, Glycobiology, 2: 25-40 (1992)).

[0080] Pharmaceutically acceptable excipients: "Pharmaceutically acceptable" excipients include any material that, when combined with the mutant FGF-21 peptide conjugates of the disclosure, retains the activity of the conjugate and is non-reactive with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical excipients such as phosphate-buffered saline, Tris buffered solution, water, salts, emulsions such as oil / water emulsions, and various types of wetting agents.

[0081] Pharmaceutical container: A "pharmaceutical container" is a container suitable for carrying a liquid pharmaceutical composition and which is generally made of an inert material and is sterile.

[0082] Administration: The term "administration" refers to oral administration, inhalation, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, or implantation of a sustained-release device (e.g., a mini-osmotic pump) into a subject. Administration is by any route, including parenteral and transmucosal (e.g., oral, nasal, vaginal, rectal or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, ventricular and intracranial. Other modes of delivery include, but are not limited to, use of liposomal formulations, intravenous infusions, transdermal patches, etc. In some embodiments, administration is subcutaneous injection.

[0083] Diabetes and diabetes-related diseases: "Diabetes" is a well-known and well-characterized disease, commonly referred to as diabetes mellitus. The term describes a group of metabolic diseases in which a person has high blood sugar levels (glycemia) because of inadequate insulin production, or because the body's cells do not respond properly to insulin, or both. Patients with high blood sugar will typically experience polyuria (frequent urination), and they will become increasingly thirsty (polydipsia) and hungry (polyphagia). "Diabetes-related diseases" are diseases characterized by the same symptoms, such as obesity, polyuria, polydipsia, and polyphagia.

[0084] Type 2 Diabetes: "Type 2 diabetes" is the most common form of diabetes / diabetes mellitus. Type 2 diabetes most commonly develops in adulthood and is more likely to occur in people who are overweight and physically inactive. Unlike type 1 diabetes, which currently cannot be prevented, many of the risk factors for type 2 diabetes can be modified. The International Diabetes Foundation lists four symptoms that signal the need for diabetes testing: a) frequent urination, b) weight loss, c) lack of energy, and d) excessive thirst. Insulin resistance is often a precursor to type 2 diabetes, a condition in which more insulin than usual is required for glucose to enter cells. Insulin resistance in the liver leads to more glucose production, while resistance in peripheral tissues means that glucose uptake is impaired.

[0085] Nonalcoholic steatohepatitis (NASH): A condition in which fat deposits in the liver, with subsequent liver damage and inflammation.

[0086] Metabolic syndrome: A defined cluster of risk factors (biochemical and physiological changes) associated with the development of type 2 diabetes and cardiovascular disease.

[0087] As used herein, "about" and "approximately" generally refer to an acceptable degree of error in a measured amount given the nature or precision of the measurement. Exemplary degrees of error are within 20 percent (%) of a given range of values, typically within 10%, and more typically within 5%. The term "substantially" refers to greater than 50%, greater than 80%, or greater than 90% or 95%.

[0088] FGF-21 peptide conjugate

[0089] Recombinant FGF-21 has been shown to affect plasma glucose and insulin levels, reduce liver and circulating triglyceride and cholesterol levels, and improve insulin sensitivity, energy expenditure, hepatic steatosis, and obesity in a range of animal models of insulin resistance. For this reason, FGF-21 is an interesting target for the treatment of human type 2 diabetes, nonalcoholic steatohepatitis (NASH), and related metabolic diseases.

[0090] Natural FGF-21 has a relatively short half-life in vivo, with reported circulating half-lives ranging from 0.5 to 4 hours in rodents and non-human primates, which limits its clinical applicability. The half-life of recombinant human FGF-21 is 1-2 hours. Various half-life extension strategies have been developed to improve the pharmacokinetic properties of FGF-21.

[0091] Abbreviations used herein include: PEG, poly(ethylene glycol); PPG, poly(propylene glycol); Ara, arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc, N-acetylgalactosamine; Glc, glucosyl; GlcNAc, N-acetylglucosamine; Man, mannosyl; ManAc, mannosamine acetate; Xyl, xylosyl; NeuAc, sialyl or N-acetylneuraminic acid; Sia, sialyl or N-acetylneuraminic acid; and derivatives and analogs thereof.

[0092] PEGylation

[0093] One method of extending the half-life of a protein is to connect one or more PEG moieties to the protein, which generally increases the biophysical solubility and stability of the protein. This method has been shown to be particularly valuable for increasing the therapeutic half-life of proteins having properties suitable for treating subjects in need thereof. However, native FGF-21 lacks specific protein PEGylation sites. Chemical PEGylation lacking specific protein PEGylation sites is not site-specific and generally results in the production of an inhomogeneous population of products that require extensive purification to achieve a uniform and highly purified product-a prerequisite for market approval of a pharmaceutical composition. Therefore, site-specific PEGylation of FGF-21 is desirable for the production of site-specific PEGylated FGF-21 peptides with improved half-life and good biological activity.

[0094] Synthesis based on enzyme has the advantages of regioselectivity and stereoselectivity. In addition, enzyme synthesis can be carried out with unprotected substrates. A possible method of PEG residue site-specifically being connected to protein is glycosyl PEGization. In glycosyl PEGization, glycosyltransferase can be used to transfer the PEG moiety to the amino acid or glycosyl residue of the amino acid connected to protein or peptide. Common final structure is protein-glycosyl moiety-optional other joint-PEG. More specifically final structure is protein-protein (N-, C- or internal) amino acid-one or more glycosyl residues-optional joint (such as amino acid joint)-straight chain or branched chain PEG moiety with different lengths, wherein glycosyl moiety can include one or more glycosyl residues. One or more glycosyl residues comprising at least a portion of the structure that protein is connected to PEG moiety can be any possible glycosyl residue. The various methods for making protein sugar PEGization are known in the art and described in detail below.

[0095] In protein PEGylation, the larger the PEG part of conjugation is, the longer the expected half-life of the protein of PEG conjugation is. This is due to the ability of the larger PEG part to protect the protein of conjugation from the impact of the protease present in the bloodstream to be relatively enhanced. Compared with smaller PEG parts, larger PEG parts give the protein of PEG conjugation a larger effective radius. Larger proteins are also more slowly degraded into the bloodstream and removed from the bloodstream than smaller proteins, because they enter the kidney more slowly or are completely blocked from entering the kidney. Therefore, the technician favors PEGylation methods, which require that the longer PEG residues of higher molecular weight (for example, ≥30kDa PEG), the PEG residues of a total higher number and / or the PEG residues of a higher degree of branching are connected to the protein, to produce a PEGylated protein with superior performance relative to the same protein conjugated with shorter / smaller PEG parts.

[0096] However, the significant disadvantage associated with PEGylation is the effectiveness of steric hindrance, whereby the conjugated PEG moiety physically blocks the active site of the protein, which is important or necessary for protein activity. For example, the PEG moiety can specifically block the receptor binding site of the protein to its receptor, which in turn leads to significant and harmful losses in protein activity. In order to avoid such potential inhibitory effects of PEGylation, those of ordinary skill in the art avoid connecting PEG near the amino acid involved in receptor binding. For FGF-21, the C-terminus is critical for β-Klotho binding, and the N-terminus is important for FGFR activation. In addition, computer simulations (insilico modeling) and in vitro efficacy determinations of FGF-21 based on the crystal structure of other FGF-21 family proteins have demonstrated that the PEGylation of amino acid residues in the putative receptor binding domain is inactive, while the PEGylation at the distal site produces the most active analogs. In addition, when the PEG moiety is placed at position 180 in FGF-21, a loss of more than 100 times of efficacy is observed in cell-based efficacy determinations. Fusion of FGF-21 to the Fc portion of an antibody has also been evaluated, with fusions at the C-terminus of FGF-21 producing much weaker analogs than fusions at the N-terminus. In contrast, N-terminally PEGylated FGF-21 has been produced and shown to be biologically active. Therefore, based on the knowledge in the art, one would avoid PEGylation close to the C-terminus of FGF-21 given the role that this region of the protein plays in binding and signaling.

[0097] Provided are methods for producing a variety of mutant fibroblast growth factor-21 (FGF-21) peptide conjugates, each comprising:

[0098] i) a mutant FGF-21 peptide comprising at least one threonine (T) residue adjacent to at least one proline (P) residue on the C-terminal side of said at least one proline residue, thereby forming at least one O-linked glycosylation site that is not present in the corresponding native FGF-21, wherein the corresponding native FGF-21 has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and

[0099] ii) 20 kDa polyethylene glycol (PEG), wherein the 20 kDa PEG is covalently linked to the mutant FGF-21 peptide at the at least one threonine residue via at least one glycosyl moiety.

[0100] In a specific embodiment, the mutant FGF-21 peptide conjugate comprises a mutant FGF-21 peptide comprising the amino acid sequence PT. In a specific embodiment, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P172T, P156T, P5T, P3T, P9T, P50T, P61T, P79T, P91T, P116T, P129T, P131T, P134T, P139T, P141T, P144T, P145T, P148T, P150T, P151T, P158T, P159T, P166T, P178T, and combinations thereof, wherein the positions of proline and threonine are based on the amino acid sequence shown in SEQ ID NO: 1. In a more specific embodiment, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of P172T, P156T, P5T and a combination thereof, specifically the group consisting of P172T, P156T and a combination thereof, wherein the positions of proline and threonine are based on the amino acid sequence shown in SEQ ID NO: 1. In a still more specific embodiment, the proline residue is located between amino acid 145 and the C-terminus of the mutant FGF-21 peptide, wherein the position of amino acid 145 is based on the amino acid sequence shown in SEQ ID NO: 1.

[0101] In another specific embodiment, the mutant FGF-21 peptide comprises the amino acid sequence P172T, wherein the positions of proline and threonine are based on the amino acid sequence shown in SEQ ID NO:1.

[0102] In another specific embodiment, the mutant FGF-21 peptide comprises mutations S173T and R176A, wherein the positions of amino acids S and R are based on the amino acid sequence shown in SEQ ID NO:1, in particular the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:2.

[0103] In another specific embodiment, the mutant FGF-21 peptide comprises the mutation Q157T, wherein the position of the amino acid Q is based on the amino acid sequence shown in SEQ ID NO:1, in particular the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:4.

[0104] In another specific embodiment, the mutant FGF-21 peptide comprises the mutation D6T, wherein the position of amino acid D is based on the amino acid sequence shown in SEQ ID NO:1, in particular the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO:5.

[0105] In other specific embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 28. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4. In some embodiments, the mutant FGF-21 peptide comprises the amino acid sequence shown in SEQ ID NO: 2. In other specific embodiments, the mutant FGF-21 peptide conjugate comprises at least one glycosyl moiety comprising N-acetylgalactosamine (GalNAc), galactose (Gal) and / or sialic acid (Sia). In a specific embodiment thereof, at least one glycosyl moiety comprises the structure -GalNAc-Sia-.

[0106] In other specific embodiments, the mutant FGF-21 peptide conjugate comprises a 20 kDa PEG moiety linked to at least one glycosyl moiety via an amino acid residue, particularly glycine (Gly). In a more specific embodiment, the mutant FGF-21 peptide conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa). Still more specifically, the mutant FGF-21 peptide conjugate comprises the following structure:

[0107]

[0108] wherein n is an integer selected from 450 to 460.

[0109] In other specific embodiments, the mutant FGF-21 peptide conjugate comprises 20 kDa PEG, which is a linear or branched PEG, particularly a linear PEG. Still more particularly, the 20 kDa PEG is a 20 kDa methoxy-PEG (see U.S. Pat. No. 10,407,479, which is incorporated herein by reference in its entirety).

[0110] Liquid pharmaceutical composition

[0111] Contemplated herein are liquid pharmaceutical compositions comprising at least one mutant FGF-21 peptide conjugate and a pharmaceutically acceptable carrier.

[0112] In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration in the range of 0.1 mg / mL to 50 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration in the range of 10 mg / mL to 48 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at a concentration of 26±4 mg / mL. For example, the FGF-21 peptide conjugate is present at a concentration of about 22, 26, 28, 30, 32, 36 mg / mL. In some embodiments, the mutant FGF-21 peptide conjugate is present at 36±6 mg / mL. For example, the FGF-21 peptide conjugate is present at a concentration of about 30, 32, 34, 36, 38, 40, 42 mg / mL.

[0113] In some embodiments, the liquid pharmaceutical composition comprises 10-48 mg / ml of the FGF-21 peptide conjugate, for example 18 mg / ml, 20 mg / ml, 28 mg / ml, 36 mg / ml, 42 mg / ml, 48 mg / ml.

[0114] Aspects of the disclosure relate to a liquid pharmaceutical composition comprising about 10 mg / ml to about 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, about 150 mM to about 500 mM arginine; about 0.01% to about 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); about 20 mM buffer, pH 7-8; and a pharmaceutically acceptable carrier, or consisting thereof. In some embodiments, the formulation has an osmotic molarity between about 250 mOsmol / kg and about 510 mOsmol / kg. In some embodiments, the liquid formulation comprises or consists of: 10 mg / ml to 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant fibroblast growth factor-21 peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first site of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between the second site of the glycosyl moiety and the 20 kDa PEG; 150 mM to 500 mM arginine; 0.01% to 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); 20 mM buffer, pH 7-8; and a pharmaceutically acceptable carrier. In some embodiments, the formulation has an osmolality of between about 250 mOsmol / kg and about 550 mOsmol / kg.In some embodiments, the liquid pharmaceutical composition comprises or consists of: about 10 mg / ml to about 48 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant fibroblast growth factor-21 peptide conjugate comprising a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO:2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG. PEG; about 150 mM to about 500 mM arginine, about 50 mM to about 250 mM alanine, about 50 mM to about 250 mM proline, about 50 mM to about 250 mM glycine, about 50 mM to about 250 mM MgCl2, about 1% to about 5% (v / v) glycerol, about 1% to 5% (v / v) PEG 400, or a combination thereof; about 0.01% to about 0.1% (w / v) polysorbate 80 (PS-80) or polysorbate 20 (PS-20); about 20 mM buffer, pH 7-8; and a pharmaceutically acceptable carrier. In some embodiments, the weight ratio of mutant FGF-21 to arginine is about 0.6 to about 0.7, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.6 to about 1, for example, about 0.6, 0.7, 0.8, 0.9, 0.1. In some embodiments, the molar ratio of mutant FGF-21 to arginine is about 0.006 to about 0.008, 0.006 to about 0.009, 0.006 to about 0.010, about 0.007 to about 0.008, about 0.007 to about 0.009, about 0.007 to about 0.010, for example, about 0.006, 0.007, 0.008, 0.009.

[0115] In some embodiments, the liquid formulation has an osmolality of about 250 mOsmol / kg to about 550 mOsmol / kg.

[0116] In some embodiments, the liquid pharmaceutical composition comprises PEG-FGF21 of 20mg / mL, arginine of 150mM, PS-80 (pH 7.5) of 0.02% (w / v) in 20mM Tris. In some embodiments, the liquid pharmaceutical formulation comprises PEG-FGF21 of 20mg / mL, arginine of 150mM, PS-80 (pH 7.5) of 0.02% (w / v) in 20mM phosphate. In some embodiments, the composition has a weight osmotic molar concentration between about 250mOsm / kg and about 380mOsm / kg. In some embodiments, the composition has a weight osmotic molar concentration of about 300mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises PEG-FGF21 of 28mg / mL, arginine of 275mM, PS-80 (pH 7-8) of 0.02% (w / v) in 20mM Tris. In some embodiments, the composition has an osmotic pressure of about 505mOsm / kg. In some embodiments, the liquid pharmaceutical formulation includes PEG-FGF21 of 18-44mg / mL, arginine of 200-350mM, PS-80 (pH 7.0-pH7.5) of 0.02% (w / v) in 20mM Tris. In some embodiments, the liquid pharmaceutical composition includes about 20mg / mL PEG-FGF21, about 150mM arginine HCl, about 20mM Tris, 0.02% (w / v) PS-80, wherein pH is about 7.5, and has an osmotic pressure of about 300mOsm / kg. In some embodiments, the liquid pharmaceutical composition includes about 28mg / mL PEG-FGF21, about 260mM arginine HCl, about 20mM Tris, about 0.02% (w / v) PS80, wherein pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL of PEG-FGF21, about 260 mM of arginine HCl, about 20 mM of Tris, about 0.02% (w / v) of PS80, wherein the pH is about 7.1, and has a weight osmotic molar concentration of about 505 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL of PEG-FGF21, about 270 mM of arginine HCl, about 20 mM of Tris, about 0.02% (w / v) of PS80, wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL of PEG-FGF21, about 270 mM of arginine HCl, about 20 mM of Tris, about 0.02% (w / v) of PS80, wherein the pH is about 7.1, and has a weight osmotic molar concentration of about 530 mOsm / kg.In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL of PEG-FGF21, 200 mM of arginine HCl, 20 mM of Tris, 0.02% (w / v) of PS80, wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL of PEG-FGF21, 200 mM of arginine HCl, 20 mM of Tris, 0.02% (w / v) of PS80, wherein the pH is about 7.1, and has an osmotic pressure of about 421 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL of PEG-FGF21, about 270 mM of arginine HCl, about 20 mM of Tris, about 0.02% (w / v) of PS80, wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL of PEG-FGF21, about 270 mM of arginine HCl, about 20 mM of Tris, about 0.02% (w / v) of PS80, wherein the pH is about 7.1, and has a weight osmotic molar concentration of about 528 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL of mutant FGF21, 200 mM of arginine HCl, 20 mM of Tris, 0.02% (w / v) of PS80, wherein the pH is 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL of PEG-FGF21, 200 mM of arginine HCl, 20 mM of Tris, 0.02% (w / v) of PS80, wherein the pH is 7.1, and has a weight osmotic molar concentration of about 455 mOsm / kg. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL PEG-FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, wherein the pH is 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL PEG-FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, wherein the pH is 7.1, and has an osmotic pressure of about 485 mOsm / kg.

[0117] In some embodiments, a response surface analysis can be performed in which arginine concentration, pH, and protein concentration can be evaluated to optimize liquid pharmaceutical compositions (see Figure 22A-22C ).

[0118] In some embodiments, the liquid composition further comprises a surfactant. In some embodiments, the surfactant comprises cetyl trimethyl ammonium bromide, sodium gluconate, or a combination thereof. In some embodiments, the liquid formulation comprises about 0.05% to about 0.1% (w / v) of cetyl trimethyl ammonium bromide, about 0.05% to about 0.1% (w / v) of sodium gluconate, or a combination thereof.

[0119] In some embodiments, liquid pharmaceutical composition also includes one or more activating agents. In some embodiments, PEG-FGF21 is co-formulated with one or more activating agents. In some embodiments, one or more activating agents may include peptides, small molecules or combinations thereof. In some embodiments, one or more activating agents may include hormones. For example, one or more reagents may include oxyntomodulin, insulin, leptin, glucagon, peroxisome proliferator-activated receptor (PPAR) agonists, FXR (farnesoid X receptor) agonists, thyroid hormone receptor-β (TRβ) agonists, sodium glucose cotransporter 2 (SGLT2) inhibitors, analogs thereof or combinations thereof. As used herein, "analogs" are molecules having modifications including one or more amino acid substitutions, deletions, inversions or additions when compared to wild-type peptide sequences.

[0120] Buffer can be present in a concentration of 1mM to 100mM. In some embodiments, buffer is present in a concentration in the range of 2mM to 75mM, 5mM to 50mM, 10mM to 25mM, 14 to 22mM. In some embodiments, buffer is present in a concentration of about 14, 16, 18, 20, 22, 24, 26, 30, 32, 34, 36, 38, 40mM or higher. For example, buffer is present in a concentration of about 20mM. pH can be in the range of 6.0 to 8.5, 6.5 to 8.0, 6.75 to 8.0, 7.1 to 8. Buffer can be a Tris phosphate buffer. For example, buffer can have a pH of 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.

[0121] Liquid pharmaceutical composition can also include tension modifier.Suitable tension modifier includes glycerol, amino acid, sodium chloride, protein or sugar and sugar alcohol.For example, modifier includes arginine, such as arginine HCl or arginine sulfate.Tension modifier exists with the concentration of 50mM to 500mM.For example, modifier (such as arginine HCL) includes arginine of 150mM to 500mM, 150mM to 275mM or 245mM to 275mM.In some embodiments, modifier includes arginine, such as arginine HCl or arginine sulfate, and it exists with the concentration between 31.6mg / ml (150mM) and 54.8mg / ml (260mM).

[0122] The liquid pharmaceutical composition may further comprise a surfactant, particularly a nonionic surfactant. The surfactant or nonionic surfactant may be a polysorbate-based nonionic surfactant, particularly polysorbate 20 or polysorbate 80, more particularly polysorbate 80.

[0123] Surfactant or nonionic surfactant can be present in a concentration of 0.01% (w / v) to 1% (w / v). For example, surfactant or nonionic surfactant can be present in a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1% (w / v).

[0124] In some embodiments, the liquid pharmaceutical composition may further comprise cetyl trimethyl ammonium bromide, sodium gluconate, or a combination thereof. For example, the composition may comprise 0.05% to 0.1% (w / v) cetyl trimethyl ammonium bromide, 0.05% to 0.1% (w / v) sodium gluconate, or a combination thereof.

[0125] In one embodiment, the liquid pharmaceutical composition comprises 10 mg / mL to 50 mg / mL of a mutant FGF-21 peptide conjugate, 1 mM to 100 mM of a buffer (e.g., Tris buffer), 150 mM to 500 mM of tensioned arginine, and 0.02% to 1% (w / v) of a polysorbate-based nonionic surfactant (particularly polysorbate 80), and has a pH of 7.0 to 8.0.

[0126] In some embodiments, the liquid formulation comprises 0.02% (w / v) PS80 (0.2 mg / ml). In some embodiments, the buffer is Tris or phosphate buffer. In some embodiments, the liquid formulation comprises 20 mM Tris buffer. In some embodiments, the liquid formulation comprises 28 mg / ml mutant FGF-21. In some embodiments, the liquid formulation comprises 36 mg / ml mutant FGF-21. In some embodiments, the liquid formulation comprises 44 mg / ml mutant FGF-21. In some embodiments, the liquid formulation comprises 150 mM to 275 mM arginine. In some embodiments, arginine is arginine HCl or arginine sulfate. In some embodiments, the pH is 7.1.

[0127] In some embodiments, the liquid pharmaceutical composition comprises about 20 mg / mL of mutant FGF21, about 150 mM arginine HCl, about 20 mM Tris, 0.02% (w / v) PS-80, and wherein the pH is about 7.5. In some embodiments, the liquid pharmaceutical composition comprises about 28 mg / mL of mutant FGF21, about 260 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 36 mg / mL of mutant FGF21, about 270 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 36 mg / mL of mutant FGF21, 200 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises about 42 mg / mL of mutant FGF21, about 270 mM arginine HCl, about 20 mM Tris, about 0.02% (w / v) PS80, and wherein the pH is about 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL of mutant FGF21, 200 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and wherein the pH is 7.1. In some embodiments, the liquid pharmaceutical composition comprises 44 mg / mL of mutant FGF21, 230 mM arginine HCl, 20 mM Tris, 0.02% (w / v) PS80, and wherein the pH is 7.1. In some embodiments, the mutant fibroblast growth factor-21 (FGF-21) peptide conjugate comprises a mutant FGF-21 peptide comprising an amino acid sequence of SEQ ID NO: 2, a glycosyl moiety, and 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO: 2 and the first site of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between the second site of the glycosyl moiety and the 20 kDa PEG. In some embodiments, the mutant FGF-21 peptide conjugate comprises the following structure:

[0128]

[0129] wherein n is an integer selected from 450 to 460.

[0130] In some embodiments, the liquid pharmaceutical composition is stable at a temperature in the range of 2-8°C for 12 months or longer. In one embodiment, the liquid pharmaceutical composition is stable at a temperature in the range of 2-8°C for up to 12 months. In one embodiment, the pharmaceutical composition is stable at room temperature for at least 3 months. In one embodiment, the pharmaceutical composition is stable at a temperature of 25°C for at least 3 months. In one embodiment, the liquid pharmaceutical composition is stable at a temperature of about -20°C for 3 months, 6 months, 12 months or more.

[0131] Also contemplated herein is a pharmaceutical container comprising a liquid pharmaceutical composition containing the same. Suitable pharmaceutical containers include, but are not limited to, syringes, autoinjectors, vials, infusion bottles, ampoules, carpoules, syringes equipped with needle protection systems, and carpoules in injection pens.

[0132] In some embodiments, the container containing the liquid pharmaceutical composition is a prefilled syringe, vial, autoinjector, or the like.

[0133] Also contemplated herein are kits comprising one or more containers containing a liquid pharmaceutical composition. Associated with the kit are instructions typically included in the commercial packaging of a therapeutic, preventive or diagnostic product, containing: information about, for example, indications, uses, dosages, manufacture, administration, contraindications and / or warnings about the use of such therapeutic, preventive or diagnostic products. The kit may also be associated with a label, which may be any type of data carrier (e.g., a leaflet, sticker, chip, print or barcode) comprising information. In certain embodiments, the instructions listed above, etc. may be included in or on a label. The kit may further include a device for administering the preparation, particularly a device containing the preparation, i.e., a pre-filled device, such as, but not limited to, a pre-filled syringe or a pre-filled automatic syringe. The kit may also include a container containing the preparation, i.e., a pre-filled container, such as a pre-filled vial, cartouche, sachet or ampoule. Also contemplated herein is a method for producing a mutant FGF-21 peptide conjugate, comprising the following steps:

[0134] (1) recombinantly producing a mutant FGF-21 peptide in an expression host; and

[0135] (2) enzymatically attaching a PEG-glycosyl moiety to the mutant FGF-21 peptide of step (1), wherein the PEG has 20 kDa, thereby forming a mutant FGF-21 peptide conjugate. In a specific embodiment, the expression host is Escherichia coli. In a more specific embodiment, step (2) includes a step (2a) of contacting the mutant FGF-21 peptide with a GalNAc donor and a GalNAc transferase under conditions suitable for transferring GalNAc from a GalNAc donor to at least one threonine residue of the mutant FGF-21 peptide. In a still more specific embodiment, the GalNAc donor is UDP-GalNAc. In another specific embodiment, the GalNAc transferase is MBP-GalNAcT2. In another specific embodiment, step (2) further comprises a step (2b) of contacting the product of step (1) or step (2a), if present, with a 20 kDa PEG-Sia donor and a sialyltransferase under conditions suitable for transferring 20 kDa PEG-Sia from the 20 kDa PEG-Sia donor to at least one threonine residue of the mutant FGF-21 peptide or to GalNAc at the mutant FGF-21 peptide if step (2a) is present. In a more specific embodiment, the 20 kDa PEG-Sia donor is 20 kDa PEG-Sia-CMP. In a more specific embodiment, the sialyltransferase is ST6GalNAc1. In a further specific embodiment, the 20 kDa PEG-Sia donor comprises the following structure:

[0136]

[0137] wherein n is an integer selected from 450 to 460.

[0138] Glycosylation and glycoconjugation methods of FGF-21 peptides

[0139] Post-expression in vitro modification of peptides and proteins is commonly used to produce glycopeptides and glycoproteins. A variety of enzymes that transfer sugar donor moieties are available, enabling in vitro enzymatic synthesis of glycoconjugates with custom-designed glycosylation patterns and glycosyl structures. See, e.g., U.S. Patent Nos. 5,876,980; 6,030,815; 5,728,554; 5,922,577; and published patent applications WO98 / 31826; WO01 / 88117; WO03 / 031464; WO03 / 046150; WO03 / 045980; WO03 / 093448; WO04 / 009838; WO05 / 089102; WO06 / 050247; WO12 / 016984; US2002 / 142370; US2003 / 040037; US2003 / 180835; US2004 / 063911; US2003 / 207406; and US2003 / 124645, each of which is incorporated herein by reference.

[0140] Due to the diversity of enzymes and methods that can be used to add and / or modify glycosyl residues on peptides, glycosyl linking groups can have basically any structure. Therefore, glycosyl linking groups can actually include any monosaccharide or oligosaccharide. Glycosyl linking groups can be connected to amino acids through side chains or through the peptide backbone. Alternatively, glycosyl linking groups can be connected to peptides through glycosyl moieties, which can be a part of the polysaccharide structure that is connected by O- on the peptide or that is connected by N-.

[0141] In accordance with the above, conjugates of glycosylated mutant FGF-21 are provided, which have glycosylation sites that are not present in the corresponding wild-type FGF-21 sequence (see U.S. Pat. No. 10,407,479, which is incorporated herein by reference in its entirety). Such conjugates are formed by enzymatically linking a modified sugar to a glycosylated FGF-21 peptide. When a modified sugar is inserted between the peptide and the modifying group on the sugar, it may be referred to herein as a "glycosyl linking group". The exquisite selectivity of enzymes (such as glycosyltransferases) is utilized to produce mutant FGF-21 peptides with desired groups at one or more specific positions. More specifically, glycosyltransferases are used to link the modified sugar to a carbohydrate moiety on the mutant FGF-21 glycopeptide.

[0142] FGF-21 conjugate

[0143] In another aspect, exemplary conjugates of modified sugars and mutant FGF-21 peptides are provided. More specifically, a mutant FGF-21 peptide conjugate comprising a mutant FGF peptide and at least one modified sugar is prepared, wherein a first of the at least one modified sugar is linked to an amino acid of the peptide via a glycosyl linking group. As described herein, the amino acid to which the glycosyl linking group is linked is mutated to generate a site recognized by a glycosyltransferase.

[0144] In another exemplary embodiment, the mutant FGF-21 peptide conjugate may comprise a mutant FGF-21 peptide and a glycosyl group linked to a mutant amino acid residue of the mutant FGF-21 peptide.

[0145] In an exemplary embodiment, the glycosyl group is a complete glycosyl linking group. In another exemplary embodiment, the glycosyl group further comprises a modifying group. In another exemplary embodiment, the modifying group is a non-glycosidic modifying group. In another exemplary embodiment, the modifying group does not include a naturally occurring sugar moiety.

[0146] Modified Sugar

[0147] In an exemplary embodiment, a mutant FGF-21 peptide is reacted with a modified sugar, thereby forming a peptide conjugate. The modified sugar comprises a "sugar donor moiety" and a "sugar transfer moiety". The sugar donor moiety is any portion of the modified sugar that will be attached to the peptide (as a conjugate described herein) through a glycosyl moiety or an amino acid moiety. The sugar donor moiety includes those atoms that are chemically altered during its conversion from the modified sugar to the glycosyl linking group of the mutant FGF-21 peptide conjugate. The sugar transfer moiety is any portion of the modified sugar that will not be attached to the peptide (as a conjugate described herein).

[0148] For the modified sugars described herein, the glycosyl moiety can be a sugar, a deoxysugar, an aminosugar, or an N-acylsugar. The term "saccharide" and its equivalents "saccharyl", "sugar" and "glycosyl" refer to monomers, dimers, oligomers, and polymers. The saccharide moiety is also functionalized with a modifying group. The modifying group is typically conjugated to the glycosyl moiety by conjugating to an amine, sulfhydryl, or hydroxyl (e.g., primary hydroxyl) moiety on the sugar. In an exemplary embodiment, the modifying group is connected through an amine moiety on the sugar (e.g., through an amide, urethane, or urea), which is formed by the reaction of an amine with a reactive derivative of the modifying group.

[0149] Any glycosyl moiety can be used as the sugar donor moiety of the modified sugar. The glycosyl moiety can be a known sugar, such as mannose, galactose or glucose, or a species with the stereochemistry of a known sugar. The general formula of these modified sugars is:

[0150]

[0151] Other glycosyl moieties that can be used in the methods described herein include, but are not limited to, fucose and sialic acid, and amino sugars such as glucosamine, galactosamine, mannosamine, 5-amine analogs of sialic acid, etc. The glycosyl moiety can be a structure found in nature, or it can be modified to provide a site for conjugating a modified group. For example, in one embodiment, the modified sugar provides a sialic acid derivative in which the 9-hydroxyl moiety is replaced by an amine. The amine is easily derivatized by an activated analog of the selected modified group. Examples of modified sugars that can be used in the methods described herein are presented in PCT Patent Application No. PCT / US05 / 002522, the entire contents of which are incorporated herein by reference.

[0152] Further exemplary embodiments utilize modified sugars in which the 6-hydroxy position is converted to a corresponding amine moiety with a linker modifying group box, such as those described above. Exemplary glycosyl groups that can be used as the core of these modified sugars include Gal, GalNAc, Glc, GlcNAc, Fuc, Xyl, Man, etc. Representative modified sugars according to this embodiment are listed below:

[0153]

[0154] Among them, R 11 -R 14 is a member independently selected from H, OH, C(O)CH3, NH and NHC(O)CH3. 10 is a linkage to, for example, another glycosyl residue (-O-glycosyl). 14 Yes OR 1 、NHR 1 or NH-LR 1 .R 1 and NH-LR 1 As described in this article.

[0155] In still further exemplary embodiments, the glycosyl group used as the core of the modified sugar in which the 6-hydroxy position is converted to the corresponding amine moiety comprises Gal and / or GalNAc.

[0156] Glycosyl linking group

[0157] In an exemplary embodiment, a mutant FGF-21 peptide conjugate comprising a modified sugar and a mutant FGF peptide described herein is presented. In this embodiment, the sugar donor portion of the modified sugar (such as a glycosyl moiety and a modifying group) becomes a "glycosyl linking group". A "glycosyl linking group" may alternatively refer to a glycosyl moiety inserted between the peptide and the modifying group.

[0158] In the following exemplary embodiments, the disclosure is illustrated by reference to selected derivatives of furanose and pyranose. It will be appreciated by those skilled in the art that the structures and compositions listed are generally applicable to the genus of glycosyl linking groups and modified sugars. Thus, the glycosyl linking group may include virtually any monosaccharide or oligosaccharide.

[0159] In an exemplary embodiment, the methods described herein utilize a glycosyl linking group having the formula:

[0160]

[0161] wherein J is a glycosyl moiety, L is a bond or linker, and R 1 is a modifying group, such as a polymer modifying group. Exemplary bonds are those formed between an NH2 moiety on the glycosyl moiety and a group having complementary reactivity on the modifying group. For example, when R 1 When a carboxylic acid moiety is included, this moiety can be activated and coupled to the NH2 moiety on the glycosyl residue to provide a moiety having the structure NHC(O)R 1 J may be an "intact" glycosyl moiety that has not been degraded by exposure to conditions that cleave pyranose or furanose structures, such as oxidative conditions, such as sodium periodate.

[0162] Exemplary linkers include alkyl and heteroalkyl moieties. Linkers include linking groups, such as acyl-based linking groups, such as -C(O)NH-, -OC(O)NH-, etc. A linking group is a bond formed between the components of the conjugate, such as between the glycosyl moiety and the linker (L), or between the linker and the modifying group (R 1 ). Other exemplary linking groups are ethers, thioethers, and amines. For example, in one embodiment, the linker is an amino acid residue, such as a glycine residue. The carboxylic acid moiety of glycine is converted into the corresponding amide by reacting with the amine on the glycosyl residue, and the amine of glycine is converted into the corresponding amide or aminoacetate by reacting with the activated carboxylic acid or carbonate of the modifying group.

[0163] NH-LR 1 An exemplary substance has the formula: NH{C(O)(CH2) a NH} s {C(O)(CH2) b(OCH2CH2) c O(CH2) d NH} t R 1 , wherein the index s and t are independently 0 or 1. The indexes a, b and d are independently integers from 0 to 20, and c is an integer from 1 to 2500. Other similar joints are based on substances in which the -NH portion is replaced by another group (e.g., -S, -O or -CH2). As understood in the art, one or more bracketed portions corresponding to the indexes s and t may be replaced by substituted or unsubstituted alkyl or heteroalkyl portions.

[0164] More specifically, the compounds described herein may include NH-L-R', wherein NH-LR' is: NHC(O)(CH2) a NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 ,NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 ,NHC(O)O(CH2) b (OCH2CH2) c O(CH2)dNHR 1 , NH(CH2) a NHC(O)(CH2) b (OCH2CH2) c O(CH2) d NHR 1 ,NHC(O)(CH2) a NHR 1 , NH(CH2) a NHR 1 and NHR 1 In these formulae, the indices a, b and d are independently selected from integers ranging from 0 to 20, for example from 1 to 5. The index c is an integer ranging from 1 to about 2500.

[0165] In an exemplary embodiment, c is selected such that the PEG moiety is about 1 kD, 5 kD, 10 kD, 15 kD, 20 kD, 25 kD, 30 kD, 35 kD, 40 kD, 45 kD, 50 kD, 55 kD, 60 kD, or 65 kD.

[0166] In a more specific embodiment, c is selected so that the PEG moiety ranges from 15-25 kD, 16-25 kD, 17-25 kD, 18-25 kD, 19-25 kD, 20-25 kD, 21-25 kD, 22-25 kD, 23-25 ​​kD, 24-25 kD, 15-20 kD, 16-20 kD, 17-20 kD, 18-20 kD, 19-20 kD, 20-30 kD, 21-30 kD, 22-30 kD, 23-30 kD, 24-30 kD, 25-30 kD, 26-30 kD, 27-30 kD, 28-30 kD, 29-30 kD. In more specific embodiments, c is selected such that the PEG moiety is 20 kD, 22 kD, 23 kD, 24 kD, 25 kD, 26 kD, 27 kD, 28 kD, 29 kD, or 30 kD.

[0167] For the purpose of clarity, the glycosyl linking group in the remainder of this portion is based on the sialic acid moiety. However, one skilled in the art will recognize that another glycosyl moiety such as mannosyl, galactosyl, glucosyl or fucosyl may be used in place of the sialic acid moiety.

[0168] In an exemplary embodiment, the glycosyl linking group is an intact glycosyl linking group, wherein the one or more glycosyl moieties forming the linking group are not degraded by chemical (e.g., sodium metaperiodate) or enzymatic (e.g., oxidase) processes. The selected conjugates of the disclosure include a modifying group attached to the amine portion of the amino-sugar, such as mannosamine, glucosamine, galactosamine, sialic acid, etc. In an exemplary embodiment, the disclosure provides a peptide conjugate comprising an intact glycosyl linking group having a formula selected from:

[0169]

[0170]

[0171] In Formula I, R 2 It is H, CH2OR 7 、COOR 7 OR 7 , where R 7 represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl. 7 When it is a carboxylic acid or a carboxylic acid ester, the two forms are separated by a single structure COO - In formula I and II, the symbol R 3 , R 4 , R 5 , R 6 and R 6 ' independently represents H, substituted or unsubstituted alkyl, OR8 、NHC(O)R 9 The index d is 0 or 1. 8 and R 9 R is independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, sialic acid or polysialic acid. 3 , R 4 , R 5 , R 6 and R 6 At least one of the R comprises a modifying group. Such a modifying group may be a polymer-modifying portion connected by a bond or a linking group, such as PEG. In an exemplary embodiment, R 6 and R 6 ' together with the carbon to which they are attached are components of the pyruvyl side chain of sialic acid. In another exemplary embodiment, the pyruvyl side chain is functionalized with a polymer modifying group. In another exemplary embodiment, R 6 and R 6 ' together with the carbon to which they are attached are components of the sialic acid side chain, and the polymer modifying group is R 5 components.

[0172] Exemplary modifying group-intact glycosyl linking group cassettes according to this motif are based on sialic acid structures, such as those having the formula:

[0173]

[0174]

[0175] In the above formula, R 1 and L are as described above. The following provides information on exemplary R 1 Further details of the structure of the group.

[0176] In yet another exemplary embodiment, the conjugate is formed between a peptide and a modified sugar, wherein the modifying group is attached to the 6-carbon position of the modified sugar via a linker. Thus, an illustrative glycosyl linking group according to this embodiment has the following formula:

[0177]

[0178] The groups are as described above. Glycosyl linking groups include, but are not limited to, glucose, glucosamine, N-acetyl-glucosamine, galactose, galactosamine, N-acetylgalactosamine, mannose, mannosamine, N-acetyl-mannosamine, and the like.

[0179] In one embodiment, the disclosure provides a mutant FGF-21 peptide conjugate comprising the following glycosyl linking group:

[0180]

[0181] Wherein D is selected from -OH and R 1 - a member of L-HN-; G is selected from H and R 1 -L- and -C(O)(C1-C6)alkyl members; R 1 is a moiety comprising a linear or branched poly(ethylene glycol) residue; and L is a linker, such as a bond ("zero order"), a substituted or unsubstituted alkyl, and a substituted or unsubstituted heteroalkyl. In an exemplary embodiment, when D is OH, G is, and when G is -C(O)(C1-C6)alkyl, D is R 1 -L-HN-.

[0182] The disclosure provides a peptide conjugate comprising a glycosyl linking group having the formula:

[0183]

[0184] In other embodiments, the glycosyl linking group has the formula:

[0185]

[0186] Here, the index t is either 0 or 1.

[0187] In yet another exemplary embodiment, the glycosyl linking group has the formula:

[0188]

[0189] Here, the index t is either 0 or 1.

[0190] In yet another embodiment, the glycosyl linking group has the formula:

[0191]

[0192] wherein the index p represents an integer from 1 to 10; and a is 0 or 1.

[0193] In an exemplary embodiment, the glycoPEGylated peptide conjugate is selected from the formula given below:

[0194]

[0195]

[0196] In the above formula, the index t is an integer from 0 to 1, and the index p is an integer from 1 to 10. Symbol R 15 ' represents H, OH (e.g. Gal- OH), sialic acid moiety, sialic acid linking group (ie, sialic acid linking group-polymer modification group (Sia-LR 1 )) or a sialic acid moiety conjugated to a polymer-modified sialic acid moiety (e.g., Sia-Sia-LR 1 )(“Sia-Sia p ”)). Exemplary polymer-modified glycosyl moieties have structures according to Formula I and II. Exemplary peptide conjugates of the disclosure will include at least one having R 15 ' polysaccharide, the polysaccharide comprising a structure according to formula I or II. The oxygen (with open valence) of formula I and II can be attached to the carbon of the Gal or GalNAc moiety via a glycosidic bond. In another exemplary embodiment, the oxygen is attached to the carbon at position 3 of the galactose residue. In an exemplary embodiment, the modified sialic acid is attached to the galactose residue on α2,3-. In another exemplary embodiment, the sialic acid is attached to the galactose residue on α2,6-.

[0197] In an exemplary embodiment, the sialic acid linking group is a sialic acid moiety, a polymer-modified sialic acid moiety (e.g., Sia-Sia-LR 1 )(“Sia-Sia p ”) is attached thereto. Here, the glycosyl linking group is attached to the galactosyl moiety via the sialic acid moiety:

[0198]

[0199] Exemplary substances according to this motif are synthesized by converting Sia-LR to Sia-LR using enzymes that form Sia-Sia bonds (e.g., CST-11, ST8Sia-II, ST8Sia-III, and ST8Sia-IV). 1 It is prepared by conjugation to the terminal sialic acid of the glycan.

[0200] In another exemplary embodiment, the glycan on the peptide conjugate has a chemical formula selected from the group consisting of:

[0201]

[0202] and combinations thereof.

[0203] In each of the above formulas, R 15 As described above. In addition, the exemplary mutant FGF-21 peptide conjugates described herein will include at least one having R 15 A polysaccharide of the moiety, the polysaccharide having a structure according to formula I or II.

[0204] In another exemplary embodiment, the glycosyl linking group includes at least one glycosyl linking group having the formula:

[0205]

[0206] Among them, R 15 is the sialyl linking group; and the index p is an integer selected from 1 to 10. In an exemplary embodiment, the glycosyl linking moiety has the following formula:

[0207]

[0208] wherein b is an integer from 0 to 1. an index s represents an integer from 1 to 10; and an index f represents an integer from 1 to 2500.

[0209] In an exemplary embodiment, the polymer modification group is PEG. In another exemplary embodiment, the PEG moiety has a molecular weight of 20-30kDa. In an exemplary embodiment, the PEG moiety has a molecular weight of 17kDa, 18kDa, 19kDa, 20kDa, 21kDa, 22kDa, 23kDa, 24kDa, 25kDa, 26kDa, 27kDa, 28kDa, 29kDa, 30kDa, 31kDa, 32kDa or 33kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 20kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of 30kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 5kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 10kDa. In another exemplary embodiment, the PEG moiety has a molecular weight of about 40kDa.

[0210] In an exemplary embodiment, the glycosyl linking group is a linear 10 kDa-PEG-sialic acid group, and one or two of these glycosyl linking groups are covalently linked to the peptide.

[0211] In an exemplary embodiment, the glycosyl linking group is a linear 20kDa-PEG-sialic acid group, and one or two of these glycosyl linking groups are covalently linked to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 30kDa-PEG-sialic acid group, and one or two of these glycosyl linking groups are covalently linked to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 5kDa-PEG-sialic acid group, and one, two or three of these glycosyl linking groups are covalently linked to the peptide. In an exemplary embodiment, the glycosyl linking group is a linear 40kDa-PEG-sialic acid group, and one or two of these glycosyl linking groups are covalently linked to the peptide.

[0212] In yet another exemplary embodiment, the mutant FGF-21 peptide is PEGylated according to the methods described herein. In a specific embodiment, the mutant FGF-21 peptide comprises a mutant S 172 T and R 176 A, wherein the positions of amino acids S and R are based on the amino acid sequence shown in SEQ ID NO: 1. More specifically, the mutant FGF-21 peptide comprises an amino acid sequence as described in SEQ ID NO: 2. As detailed above, the at least one glycosyl moiety that is linked to the threonine residue and that links the newly introduced threonine residue to the PEG portion can be virtually any possible glycosyl moiety. The only restrictions are that it should be able to be linked to threonine and that it should be able to be linked to PEG or m-PEG, more particularly through a linker, such as an amino acid residue, particularly glycine. In a specific embodiment, at least one glycosyl moiety comprises N-acetylgalactosamine (GalNAc), galactose (Gal) and / or sialic acid (Sia). In a more specific embodiment, at least one glycosyl moiety comprises the structure -GalNAc-Sia-, i.e. two glycosyl moieties, i.e. GalNAc and Sia, wherein the PEG residue can be linked to GalNAc or Sia, particularly to Sia. The glycosyl moiety that is not linked to the PEG portion can be linked to the newly introduced threonine residue.

[0213] In another specific embodiment, the 20 kDa PEG moiety is linked to at least one glycosyl linker via a linker, such as an amino acid residue, particularly a small amino acid such as alanine or glycine, more particularly via glycine (Gly). Thus, the PEG or m-PEG moiety is linked to the amino acid and the amino acid is linked to the glycosyl moiety such as Sia. The glycosyl moiety is linked to the amino acid linker (if present) and to the newly introduced threonine residue in the mutant FGF-21 amino acid sequence. The amino acid residue is linked to the PEG and glycosyl residue by the method described in WO03 / 031464, which is incorporated herein by reference.

[0214] In a specific embodiment, the mutant FGF-21 peptide (e.g., SEQ ID NO: 2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa), wherein GalNAc is linked to, for example, a newly introduced threonine residue and to Sia. Sia is further linked to a PEG of 17 kDa, 18 kDa, 19 kDa, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32 kDa or 33 kDa via a glycine residue.

[0215] In a more specific embodiment, the mutant FGF-21 peptide (e.g., SEQ ID NO: 2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa), wherein GalNAc is linked to, for example, a newly introduced threonine residue and to Sia. Sia is further linked to PEG of 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa or 30 kDa via a glycine residue.

[0216] In still more specific embodiments, the mutant FGF-21 peptide (e.g., SEQ ID NO: 2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa), wherein GalNAc is linked to, for example, a newly introduced threonine residue and to Sia. Sia is further linked to a PEG of 20 kDa, 25 kDa or 30 kDa via a glycine residue.

[0217] In another specific embodiment, the mutant FGF-21 peptide (e.g., SEQ ID NO: 2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa), wherein GalNAc is linked to, for example, a newly introduced threonine residue and to Sia. Sia is further linked to a 20 kDa or 30 kDa PEG via a glycine residue.

[0218] In another specific embodiment, the mutant FGF-21 peptide (e.g., SEQ ID NO: 2) conjugate comprises the structure -GalNAc-Sia-Gly-PEG (20 kDa), wherein GalNAc is linked to, for example, a newly introduced threonine residue and to Sia. Sia is further linked to a 20 kDa PEG via a glycine residue.

[0219] In a very specific embodiment, the mutant FGF-21 peptide conjugate comprises the following structure:

[0220]

[0221] Wherein, n is an integer selected from 450 to 460, and similarly Figure 1 shown.

[0222] 20kDa PEG can be straight or branched, more specifically, 20kDa PEG is a straight 20kDa PEG. In addition, 20kDa PEG is specifically 20kDa methoxy-PEG (mPEG, m-PEG). PEG and mPEG of different molecular weights can be obtained from various suppliers, such as from Jenkem Technology USA, Plano, TX, USA or Merckle Biotec, Ulm, Germany. It should be understood in the art that PEG 20kDa refers to that the size of the PEG residue is 20kDa on average, and the size of most of the PEG residues is 20kDa.

[0223] Mutant FGF-21 peptides and conjugates thereof

[0224] Provided are variants of fibroblast growth factor-21 (FGF-21) having unexpected properties, including variants having unusually long half-lives, which are peptide conjugates comprising:

[0225] i) a mutant FGF-21 peptide comprising at least one threonine (T) residue adjacent to at least one proline (P) residue on the C-terminal side of at least one proline residue, thereby forming at least one O-linked glycosylation site that is not present in the corresponding native FGF-21, wherein the corresponding native FGF-21 has an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, and

[0226] ii) 20-30 kDa polyethylene glycol (PEG), wherein the 20-30 kDa PEG is covalently linked to the mutant FGF-21 peptide at at least one threonine residue via at least one glycosyl moiety.

[0227] For the attachment of a 20-30 kDa PEG residue, a threonine residue is introduced into the amino acid sequence of native FGF-21 adjacent to and on the C-terminal side of a proline residue already present in the amino acid sequence of native FGF-21 (i.e., a native proline residue). For this purpose, (i) an additional threonine residue may be introduced adjacent to the native proline residue, or (ii) a native amino acid present in the native amino acid sequence of FGF-21 adjacent to and located on the C-terminal side of the native proline residue is replaced with a threonine residue. In the disclosure, option (ii) is an exemplary embodiment. As described herein, one or more threonine residues may be introduced adjacent to and at the C-terminus of an already existing proline residue. Thus, the mutant FGF-21 of the disclosure may contain both a threonine residue that has been additionally introduced and a threonine residue that has been introduced in place of a native amino acid.

[0228] By introducing a new threonine residue on the C-terminal side and adjacent to a proline residue, a consensus sequence for O-glycosylation enzymes was formed. Because proline residues are typically found on the surface of proteins (e.g., in turns, kinks, and / or loops), it is desirable to use a PEG-sugar moiety immediately adjacent to the proline residue for its O-glycosylation and the design of PEGylation benefits from the relative accessibility of the target attachment site for glycosyltransferases to transfer the glycosyl or diol-PEG moiety and the potential to employ conjugated glycosyl and / or PEG structures without disrupting the protein structure.

[0229] To introduce threonine residues into the native amino acid sequence of FGF-21, conventional techniques in the field of recombinant genetics are used. Basic texts that disclose the general methods used in the published texts include Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd edition, 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology (1994).

[0230] In a specific embodiment, the native FGF-21 amino acid sequence corresponds to the native amino acid sequence of human FGF-21 depicted in SEQ ID NO:1.

[0231] In a specific embodiment, the mutant FGF-21 peptide comprises the amino acid sequence PT, i.e., a threonine residue adjacent to the C-terminus of a proline residue. The sequence PT does not exist in the native FGF-21 amino acid sequence.

[0232] Optionally, the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of: 172 T (e.g., SEQ ID NO: 2 or 3), P 156 T (e.g., SEQ ID NO: 4), P 5 T (e.g., SEQ ID NO: 5), P 3 T (e.g., SEQ ID NO: 6), P 9 T (e.g., SEQ ID NO: 7), P 50 T (e.g., SEQ ID NO: 8), P 61 T (e.g., SEQ ID NO: 9), P 79 T (e.g., SEQ ID NO: 10), P 91 T (e.g., SEQ ID NO: 11), P 116 T (e.g., SEQ ID NO: 12), P 120T (e.g., SEQ ID NO: 13), P 125 T (e.g., SEQ ID NO: 14), P 129 T (e.g., SEQ ID NO: 15), P 131 T (e.g., SEQ ID NO: 16), P 134 T (e.g., SEQ ID NO: 17), P 139 T (e.g., SEQ ID NO: 18), P 141 T (e.g., SEQ ID NO: 19), P 144 T (e.g., SEQ ID NO: 20, P 145 T (e.g., SEQ ID NO: 21), P 148 T (e.g., SEQ ID NO: 22), P 150 T (e.g., SEQ ID NO: 23), P 151 T (e.g., SEQ ID NO: 24), P 158 T (e.g., SEQ ID NO: 25), P 159 T (e.g., SEQ ID NO: 26), P 166 T (e.g., SEQ ID NO: 27), P 178 T (e.g., SEQ ID NO: 28) and combinations thereof, wherein the positions of proline and threonine are based on the natural amino acid sequence of FGF-21 as described in SEQ ID NO: 1, specifically the mutant FGF-21 peptide comprises at least one amino acid sequence selected from the group consisting of: P 172 T.P 156 T.P 5 T and combinations thereof, more specifically P 172 T.P 156 T and combinations thereof, and even more specifically, the mutant FGF-21 peptide comprises a sequence motif P based on the amino acid sequence as set forth in SEQ ID NO: 1 172 T, wherein the positions of proline and threonine are based on the amino acid sequence described in SEQ ID NO:1.

[0233] In a specific embodiment, the proline residue is located between amino acid 145 and the C-terminus of the mutant FGF-21 peptide, wherein the position of amino acid 145 is based on the amino acid sequence described in SEQ ID NO: 1. As demonstrated by the results presented herein, the C-terminus of FGF-21 is surprisingly tolerant to the attachment of PEG and in particular glycosyl-PEG moieties. This is unexpected because the literature reports that an intact C-terminus is necessary for β-Klotho binding of FGF-21.

[0234] In a specific embodiment, the mutant FGF-21 peptide comprises a mutation S 172 T and R 176 A, wherein the positions of amino acids S and R are based on the amino acid sequence described in SEQ ID NO: 1, in particular the mutant FGF-21 peptide comprises the amino acid sequence described in SEQ ID NO: 2. It has been found that the mutant R 176 A is beneficial to the overall stability of the protein after the introduction of an O-linked glycosylation site at threonine 173. By this mutation, the relatively large arginine side chain is removed and replaced with the small side chain of alanine. It is assumed that the smaller side chain of alanine interferes less with the large number of glycosyl-PEG moieties to be attached to the indicated (thindicae) mutant FGF-21 peptide. In an alternative embodiment, the mutant FGF-21 peptide comprises the mutation Q 157 T, wherein the position of amino acid Q is based on the amino acid sequence described in SEQ ID NO: 1, in particular, the mutant FGF-21 peptide comprises the amino acid sequence described in SEQ ID NO: 4, or a mutant D 6 T, wherein the position of amino acid D is based on the amino acid sequence described in SEQ ID NO:1, in particular the mutant FGF-21 peptide comprises the amino acid sequence described in SEQ ID NO:5.

[0235] In a specific embodiment, the mutant FGF-21 peptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 28. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. In some embodiments, the mutant FGF-21 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.

[0236] In some embodiments, the mutant FGF-21 peptide comprises the amino acid sequence depicted in SEQ ID NO:2.

[0237] Further provided is a liquid pharmaceutical composition comprising a mutant FGF-21 peptide conjugate and a pharmaceutically acceptable carrier such as water or a physiologically compatible buffer. The liquid pharmaceutical composition generally comprises a therapeutically effective amount or a pharmaceutically active amount of a mutant FGF-21 peptide conjugate as an active agent.

[0238] In some embodiments, the liquid pharmaceutical composition further comprises one or more active agents. In some embodiments, one or more agents may include peptides, small molecules, or combinations thereof. In some embodiments, one or more agents may include hormones. For example, one or more agents may include oxyntomodulin, insulin, leptin, glucagon, peroxisome proliferator-activated receptor (PPAR) agonists, FXR (farnesoid X receptor) agonists, thyroid hormone receptor-β (TKβ) agonists, sodium glucose cotransporter 2 (SGLT2) inhibitors, analogs thereof, or combinations thereof.

[0239] The liquid pharmaceutical composition of the disclosure is suitable for use in a variety of drug delivery systems. Suitable formulations for the disclosure are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pennsylvania, 17th edition (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990). The pharmaceutical composition is intended for parenteral, intranasal, topical, oral or local administration, such as by subcutaneous injection, aerosol inhalation or transdermal adsorption, for preventive and / or therapeutic treatment. In some embodiments, the liquid pharmaceutical composition is administered by injection. In some embodiments, the liquid pharmaceutical composition is administered parenterally (e.g., subcutaneously or intravenously). According to various aspects of the disclosure, the liquid pharmaceutical composition is administered by subcutaneous injection. Therefore, aspects of the disclosure provide a composition for parenteral administration (e.g., subcutaneous injection) comprising a mutant FGF-21 peptide conjugate dissolved or suspended in an acceptable carrier. The carrier can be an aqueous carrier, such as water, buffered water, saline, phosphate buffered saline (PBS), etc. The composition may also contain detergents such as Tween 20 and Tween 80; stabilizers such as mannitol, sorbitol, sucrose and trehalose; and preservatives such as EDTA and m-cresol. The composition may contain pharmaceutically acceptable auxiliary substances required to approach physiological conditions, such as pH adjustment and buffering agents, tonicity adjusting agents, wetting agents, detergents, etc.

[0240] The liquid pharmaceutical composition of the disclosure can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier before administration. The composition comprising the FGF peptide conjugate can be used for preventive and / or therapeutic treatment, particularly for the treatment of diabetes or diabetes-related diseases, particularly for the treatment of type 2 diabetes, NASH and metabolic syndrome. In therapeutic applications, the composition is administered to a subject who has already suffered from a disease or condition related to diabetes in an amount sufficient to cure or at least partially prevent the symptoms of the disease and its complications. An amount sufficient to achieve this is defined as a "therapeutically effective amount" and is generally dependent on the patient's health and weight.

[0241] The disclosure provides a method for treating a disease and / or condition or its symptoms, the method comprising administering a liquid pharmaceutical composition comprising them to a subject (e.g., a mammal, such as a human). Therefore, one embodiment is a method for treating a subject suffering from diabetes or a diabetes-related disease (e.g., type 2 diabetes, NASH, or metabolic syndrome) or its symptoms. The method comprises the following steps: administering to a mammal a certain amount of a compound described herein or a composition comprising the compound under conditions of treating a disease or condition, the amount being sufficient to treat a disease or condition or its symptoms.

[0242] Single or multiple administrations of the liquid composition may be carried out with a dosage level and pattern selected by the treating physician. In any case, the liquid pharmaceutical composition should provide an amount of the mutant FGF-21 peptide conjugate of the disclosure sufficient to effectively treat a subject in need of such treatment.

[0243] In another aspect, a method for treating diabetes and related diseases, particularly type 2 diabetes, non-alcoholic steatohepatitis (NASH) and / or metabolic syndrome is provided, the method comprising administering a liquid pharmaceutical composition to a subject in need thereof, the liquid pharmaceutical composition comprising at least one of the mutant FGF-21 peptide conjugates described herein. In a specific embodiment, the subject in need thereof is a human subject.

[0244] In liquid pharmaceutical compositions, the mutant FGF-21 peptide conjugate is typically present in a concentration ranging from about 0.1 mg / mL to about 50 mg / mL, for example, from about 10 mg / mL to about 48 mg / mL, from about 20 mg / mL to about 44 mg / mL, for example, about 20 mg / mL, about 28 mg / mL, about 36 mg / mL, about 44 mg / mL.

[0245] All components of the liquid pharmaceutical composition, as well as the specific concentrations of the components, have been carefully selected after testing a very large number of different conditions, compounds and their concentrations. Therefore, the pharmaceutical compositions disclosed herein are not an arbitrary selection of compounds and compound concentrations, but rather a specific and rational selection of conditions that were found to be optimal for aqueous pharmaceutical compositions comprising mutant FGF-21 peptide conjugates or mutant FGF-21 peptides according to the disclosure for use as a medicament.

[0246] In some embodiments, the liquid pharmaceutical composition comprises a buffer, particularly phosphate or Tris buffer, such as tris (hydroxymethyl) aminomethane (THAM). Optionally, the buffer is present in a concentration of 1mM to 100mM, 2mM to 75mM, 5mM to 50mM, 10mM to 25mM. Select 5 to 25mM Tris buffer because it is found that the solubility of the protein is better than other buffer systems, and is suitable for maintaining pH at 7-8, such as pH 7.5+ / -0.4 (e.g. 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9). In some embodiments, the liquid pharmaceutical composition comprises about 20mM Tris buffer. For the extended storage of the mutant FGF-21 peptide conjugates of PEGylation, the pH seems to be optimal. In addition, the probability of Tris crystallization at lower temperatures is lower than that of phosphate-based buffers.

[0247] In some embodiments, the pH of the pharmaceutical composition is in the range of 6.0 to 8.5, 6.5 to 8.0, 6.75 to 8.0, 7.0 to 8.0, 7.0 to 8.0, e.g., 7.5 ± 0.5, because if the pH is in the range of 7-8, the lowest fragmentation of SDS-PAGE and the least aggregation of SEC are observed. It is also considered that the pH is optional with respect to viscosity. Because the pH of a solution can depend on the temperature of the solution, the pH should be adjusted and measured, in particular, at 25 ± 2 ° C. The pH can be adjusted with HCl.

[0248] The pharmaceutical composition may further comprise a tonicity modifier. The tonicity modifier may be selected from the group consisting of glycerol, amino acids, sodium chloride, proteins, sugars and sugar alcohols. It was found that tonicity modifiers, particularly arginine, have a beneficial effect on the pharmaceutical composition because it reduces the aggregation of the active agent, i.e. the mutant FGF-21 peptide (conjugate).

[0249] The tonicity modifier (e.g., arginine) can be present at a concentration of about 150 mM to about 500 mM, about 150 mM to about 275 mM, about 220 mM to about 270 mM, about 200 mM to about 260 mM, about 200 mM to about 230 mM. For example, arginine HCl can be present at a concentration of about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, or 270 mM.

[0250] In addition, pharmaceutical compositions can include surfactants, particularly nonionic surfactants. Surfactants or nonionic surfactants are particularly nonionic surfactants based on polysorbate. In some embodiments, surfactant is polysorbate 20 or polysorbate 80. The surfactant or nonionic surfactant (for example polysorbate 20 or 80) is optionally present in a concentration of 0.01mg / mL to 1mg / mL, 0.05 to 0.5mg / mL or 0.2 ± 0.02mg / mL. In some embodiments, surfactant is polysorbate 80. In some embodiments, surfactant is present in a concentration of 0.2 ± 0.02mg / mL. For example, surfactant can be present in a concentration of about 0.18, 0.19, 0.2, 0.21, 0.22mg / mL. In some embodiments, surfactant, such as nonionic surfactant based on polysorbate, prevents the shear force that can cause aggregation.

[0251] Also provided herein is a pharmaceutical container comprising a liquid pharmaceutical composition of the disclosure. In some embodiments, the pharmaceutical container is a syringe, an autoinjector, a vial, an infusion bottle, an ampoule, a carpoule, a syringe equipped with a needle protection system, or a carpoule in an injection pen.

[0252] manufacture

[0253] In some embodiments, the method is as follows: First, mutations that introduce threonine adjacent to the C-terminal side of the proline residue and thereon, and optionally one or more additional mutations, are introduced into a nucleic acid sequence encoding a native or mutant FGF-21 (such as human FGF-21 shown in SEQ ID NO: 1). The nucleic acid sequence encoding the mutant FGF-21 peptide is introduced into an expression vector suitable for expressing the protein in an expression host. Methods for introducing mutations into nucleic acid sequences, such as site-directed mutagenesis, and incorporating the mutant nucleic acid sequence into an expression vector are well known to the skilled person (see, for example, RB Corley, "A Guide to Methods in the Biomedical Sciences", Springer Science & Business Media, 2006).

[0254] After protein expression, optional purification, a PEG residue is linked to the mutant FGF-21 peptide via at least one glycosyl moiety and optionally via at least one amino acid residue present between the PEG and the glycosyl residue, specifically at the newly introduced threonine residue.

[0255] In order to obtain high yield expression of the nucleic acid encoding the mutant FGF-21 of the disclosure, the polynucleotide encoding the mutant fibroblast growth factor is typically subcloned into an expression vector containing a strong promoter to direct transcription, a transcription / translation terminator, and a ribosome binding site for translation initiation. Suitable bacterial promoters are well known in the art and are described, for example, in Sambrook and Russell (supra) and Ausubel et al. (supra). Bacterial expression systems for expressing native or mutant FGF-21 are available, for example, in Escherichia coli (E. coli), Bacillus sp., Salmonella, and Caulobacter. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated vector, or a retroviral vector. In some embodiments, the mutant FGF-21 peptide is recombinantly produced in E. coli cells, ie, the expression host is E. coli.

[0256] An exemplary production method is described in this paragraph: The mutant FGF-21 peptide is expressed as inclusion bodies in E. coli. The cells are recovered from the harvest by centrifugation, disrupted, and the inclusion bodies are washed and recovered by centrifugation. The purification of the non-PEGylated mutant FGF-21 peptide begins with solubilizing the mutant FGF-21 peptide from the inclusion bodies and refolding the peptide. The refolded mutant FGF-21 peptide is filtered and purified by two anion exchange chromatography operations, both utilizing Eshmuno Q chromatography resin and operating in bind and elute mode. If necessary, the purified mutant FGF-21 peptide can be concentrated by ultrafiltration using a Pellicon2 (5kD MWCO) membrane. The purified mutant FGF-21 peptide is dispensed into sterile PETG bottles and can be stored at ≤70°C. See Figure 2 and Figure 3 .

[0257] GlycoPEGylation of mutant FGF-21 peptides can be performed by two enzymatic reactions in series or simultaneously. In the process where the enzymatic steps are in series, an anion exchange chromatography step (e.g., an anion exchange chromatography step) can be performed between two consecutive enzymatic steps. This step can be followed by 0.2 μm filtration and two anion exchange chromatography operations, both of which utilize Q Sepharose Fast Flow chromatography resins and operate in bind and elute mode. The final concentration step can be performed by ultrafiltration using Pellicon XL Biomax (10 kDa MWCO). Figures 2 to 4 A non-limiting exemplary production method is shown. Figure 4 . Two main classes of enzymes are used in the synthesis of carbohydrates, glycosyltransferases (e.g., sialyltransferases, oligosaccharyltransferases, N-acetylglucosaminyltransferases) and glycosidases. Glycosidases are further classified into exoglycosidases (e.g., β-mannosidases, β-glucosidases) and endoglycosidases (e.g., endoglycosidase-A, endoglycosidase-M). Each of these classes of enzymes has been successfully synthesized for the preparation of carbohydrates. For a general review, see Crout et al., Curr. Opin. Chem. Biol. 2: 98-111 (1998). See also PCT Publication Nos. WO2003 / 031464; WO2005 / 089102; WO2006 / 050247; and WO2012 / 016984, each of which is incorporated herein by reference in its entirety.

[0258] In some embodiments, the 20 kDa PEG-Sia donor comprises the structure

[0259]

[0260] Wherein, n is an integer selected from 450 to 460, which produces a molecular weight of 20 kDa. The structure includes a Gly linker. The skilled person understands that any method for producing it can be used, such as the method described in PCT Publication No. WO2003 / 031464, the entire contents of which are incorporated herein by reference.

[0261] In some embodiments, the FGF-21 peptide conjugate is filtered using a 0.22 micron filter. Additionally, the mutant FGF-21 peptide conjugate can be sterile filtered.

[0262] Also provided are mutant FGF-21 peptide conjugates obtainable by the methods of the disclosure.

[0263] In some embodiments, the concentrated protein solution (PEG FGF-21) is thawed from -80C until liquid. In some embodiments, buffer exchange is carried out by diafiltration process, wherein about 7 volumes are exchanged with formulation buffer, 20mM Tris, 150mM arginine HCl, pH7.5. In some embodiments, diafiltration buffer is used to adjust protein concentration to about 20mg / mL. In some embodiments, polysorbate 80 (PS80) is then added to 0.02%. In some embodiments, 0.22 μm filter is used to aseptically filter the solution and fill into a sterile bottle.

[0264] application

[0265] The disclosure provides a liquid pharmaceutical composition, which is used as a medicine and is used to treat diabetes and related diseases, particularly type 2 diabetes, non-alcoholic steatohepatitis (NASH) and / or metabolic syndrome. The disclosure also provides the use of the liquid pharmaceutical composition for treating diabetes and related diseases, particularly type 2 diabetes, NASH and / or metabolic syndrome.

[0266] Further provided is a method of treating diabetes and related diseases, in particular type 2 diabetes, NASH, non-alcoholic fatty liver disease (NAFLD) and / or metabolic syndrome, the method comprising administering a liquid pharmaceutical composition according to the disclosure to a subject in need thereof. In a specific embodiment, the subject is a human subject.

[0267] NAFLD is a common chronic liver disease in Western countries that can progress to cirrhosis and is often associated with an increased risk of mortality, particularly cardiovascular mortality. Current pharmacological treatments for NAFLD have limited efficacy, and there is an urgent need to develop more effective and safe agents for this common and life-threatening disease. Obeticholic acid (OCA), a selective agonist of the farnesoid X receptor, appears promising as a therapeutic agent for the management of NAFLD. The Farnesoid X Receptor Ligand Obeticholic Acid in NASH Treatment (FLINT) trial in patients with NASH revealed that OCA administration was associated with improvements in liver histology as well as weight loss and reductions in blood pressure. Although the adverse effects of OCA on lipid profiles and insulin sensitivity are noteworthy, OCA may be considered in selected patients with NAFLD / NASH, especially those with adequate control of glucose and lipid levels.

[0268] About the index of clinical efficacy of the compounds and compositions described herein, multiple exemplary indexes are known in the art and described herein, including but not limited to HbA1c, glucose and insulin, body weight, serum lipids (total cholesterol, LDL, triglycerides), liver enzymes (ALT, AST), liver weight, relative liver weight (% body weight), NAFLD activity score (NAS), fibrosis score (e.g., liver fibrosis), proinflammatory cytokines (e.g., IL1β, MCP-1), fibrosis biomarkers (αSMA, collagen 1α), liver cholesterol, liver triglycerides and liver fatty acid decline. The increase of at least one of high molecular weight (HMW) adiponectin or HDL is also an index of clinical efficacy of compounds and compositions described herein. Therefore, the change of at least one index (as noted above) reflects the clinical efficacy of compounds or compositions described herein.

[0269] In some embodiments, the therapeutic efficacy of a compound or composition described herein is determined based on a decrease in at least one of serum triglyceride levels or serum insulin levels.

[0270] For example, HOMA-IR is an indicator of the presence and degree of insulin resistance in a subject. It is an accurate indicator of the dynamics between baseline (fasting) blood glucose and the insulin level in response to it. It is called an insulin resistance calculator. For humans, the healthy range is 1.0 (0.5-1.4). Less than 1.0 indicates that the subject is insulin sensitive, which is ideal; higher than 1.9 indicates that the subject exhibits early insulin resistance; higher than 2.9 indicates that the subject exhibits significant insulin resistance. The HOMA-IR blood code calculation is determined as follows: Insulin uIU / mL (mU / L) X glucose (mg / dL) = HOMA-IR. This calculation requires American standard units. In order to convert from international SI units: for insulin: pmol / L to uIU / mL, divide by (÷) 6; for glucose: mmol / L to mg / dL, multiply by (X) 8.

[0271] Also presented herein is a treatment regimen whereby a liquid pharmaceutical composition comprising a therapeutically effective amount of a mutant FGF-21 peptide conjugate is administered twice a day, once a day, every two days, three times a week, once a week, once every two weeks, once every three weeks, or once a month. For example, a liquid pharmaceutical composition comprising about 10 mg to up to 45 mg, about 20 mg to about 44 mg, about 28 mg to about 44 mg, about 36 mg to about 44 mg, about 20 mg to about 36 mg, about 28 mg to about 36 mg, about 20 mg to about 28 mg of a mutant FGF-21 peptide conjugate (e.g., about 10 mg, 18 mg, 20 mg, 25 mg, 28 mg, 30 mg, 36 mg, 42 mg, or 44 mg) can be administered to a subject in need thereof twice a day, once a day, every two days, three times a week, once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, a liquid pharmaceutical composition comprising up to 44 mg / ml of a mutant FGF-21 peptide conjugate is administered to a subject in need thereof once a week or once every two weeks. The long-duration efficacy of the mutant FGF-21 peptide conjugates described herein is demonstrated by the unexpectedly long half-life of these conjugates measured in an animal model system. The long-duration efficacy of the mutant FGF-21 peptide conjugates described herein in turn allows for less frequent administration of the mutant FGF-21 peptide conjugates. Therefore, in some embodiments, the mutant FGF-21 peptide conjugates described herein or compositions comprising the same are administered to a subject in need thereof at a frequency equal to or greater than once a week. For example, the mutant FGF-21 peptide conjugate described herein or a composition comprising the same can be administered to a subject in need thereof once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, once every 20 days, once every 21 days, once every 22 days, once every 22 days, once every 23 days, once every 24 days, once every 25 days, once every 26 days, once every 27 days, once every 28 days, once every 29 days, once every 30 days, or once every 31 days.

[0272] In another exemplary treatment regimen, the compounds described herein and compositions comprising the same follow an "induction" course of treatment, which requires more frequent administration, such as twice a week or once a week at the beginning of the treatment regimen, followed by maintenance therapy, which may involve administration once a week or once a month. Such a regimen is effective because the initial induction therapy improves the subject's condition to a manageable level that is acceptable for achieving a clinical state that is acceptable for maintenance of the disease / disorder. Thereafter, maintenance therapy is used to maintain the health level at a maintenance level.

[0273] The therapeutic efficacy of the compound and / or composition for treating diabetes and related diseases, particularly type 2 diabetes, nonalcoholic steatohepatitis (NASH) and / or metabolic syndrome can be evaluated using various parameters and assays known to those skilled in the art and described herein. Measuring hemoglobin A1c (HbA1C) is considered to be a standard assay for measuring the glycemic index of a subject in a long-duration period. Therefore, it is a stable index of glycemic index, reflecting the glucose level in the process of approximately nearly 3-4 months. Therefore, the subject suffering from diabetes (such as type 2 diabetes) can be limited by the percentage HbA1C determined in a suitable assay.

[0274] For a healthy person without diabetes, the normal range of hemoglobin A1c levels is between 4% and 5.6%. A hemoglobin A1c level between 5.7% and 6.4% indicates that the person has a higher chance of developing diabetes. A level of 6.5% or higher indicates that the person has diabetes.

[0275] In some embodiments, HbA1C is measured by HPLC using a glycosylated hemoglobin test system (BIO-RAD, Hercules, CA, USA). Blood samples (e.g., 1.0 mL / time) can be collected from the cephalic vein or saphenous vein to the BD K2-EDTA tubes. The samples can be stored immediately at 4°C or kept on wet ice and analyzed on the same day of blood collection. The HbA1c level in the blood can be measured by HPLC using a glycosylated hemoglobin test system (BIO-RAD, Hercules, California, the United States) by those skilled in the art.

[0276] Regarding NASH, the condition is currently only diagnosed by biopsy. However, there are some abnormal biomarkers that are considered predictors of NASH, such as liver fat (determined by MRI), liver enzymes (ALT and ALT / AST ratio), and fibrosis biomarkers (such as pro-C3).

[0277] Example

[0278] The following examples illustrate specific embodiments of the disclosure. They are set forth for illustrative purposes only, and they should not be construed as limiting the disclosure.

[0279] Liquid preparations

[0280] A liquid formulation of a glycoPEGylated FGF-21 mutant (PEG-FGF21, also referred to herein as a mutant FGF-21 peptide conjugate) was developed. The formulation is supported by stability data for up to 12 months stored at 2-8°C and is suitable for clinical or home administration. Additional optimization was performed to produce an improved formulation with a higher PEG-FGF21 concentration.

[0281] Pre-formulation development studies of PEG-FGF21 were conducted to establish a formulation composition that would provide optimized physical, chemical, and structural stability for the protein. Various buffers, excipients, surfactants, and pH were screened to optimize protein stability at a target concentration of approximately 20 mg / mL.

[0282] Based on baseline biophysical screening I (BBSI), screening has a series of buffers with different pH to test sixteen (16) unique formulations, to identify suitable pH / buffer combinations by testing PEGylated and non-PEG-FGF21 with 2mg / mL.Test samples by static light scattering (SLS) and dynamic light scattering (DLS).DSF results for non-PEG-FGF21 and DLS and SLS results for both PEGylated and non-PEGylated materials show that the pH range of 6.5 to 8.5 is suitable for protein solubility.In a second study, baseline biophysical screening II (BBSII) was assessed, the effect of the NaCl of 150mM, the arginine-HCl of 150mM, the sucrose of 250mM and the sorbitol of 250mM as excipients in buffers within the range of pH 6.5 to 8.0.SLS, DLS and DSF are carried out with 2mg / mL on PEGylated and non-PEG-FGF21. Providing Tris at pH 8.0 and phosphate at pH 7.5 with arginine, sucrose, and sorbitol is best for PEG-FGF21 and non-PEG-FGF21 stability ( Figure 5-Figure 11 ).

[0283] In the 3rd research-solubility study, the optimum combination of pH, buffer and excipient set up in BBSII is used to determine that under high concentration (about 50mg / mL), PEG-FGF21 is provided with the preparation of stability. Sample is carried out DLS and SEC test, and determines that phosphate / arginine-HCl and phosphate / sucrose preparation produce preparation / SEC analysis as low aggregate, and also shows the DLS curve with lower intensity for large aggregate. Selecting Tris / sucrose is because it has the total aggregate of minimum percentage by SEC, and owing to having the low aggregate as indicated by SEC and DLS and selecting Tris / sorbitol and Tris / arginine-HCl. The 4th research determines the influence of surfactant on product stability and finds that product is harmless.

[0284] The results obtained by the first four studies are used to select the design space from the experimental study design (DOE). The randomized response surface design with two numerical factors (pH and protein concentration), two categorical factors (buffer and excipient type) is used to develop a design with six center points. A total of 36 preparations (tables 1 and 2) are selected to screen PEG-FGF21 and to add four other non-DOE (off-DOE) preparations to screen the combination of arginine and gluconate and cetyl trimethylammonium bromide.

[0285] Table 1. Selected formulations (DS design for experimental study) from optimization schemes of rounds 1 and 2

[0286]

[0287]

[0288]

[0289] The resulting samples were stored at 5°C for 8 weeks and at 25°C for 2 weeks and tested by UV, SEC, RP-HPLC and DLS. Viscosity. Microflow imaging (MFI), DSF and pH tests were performed only at the initial time point (T=0). DLS tests were performed only at week 2 (2W). The data showed that dimerization was effectively controlled by increasing arginine concentration ( Fig.21 ). Figure 11-Figure 16 The key results of the DOE are presented in. The results provide a formulation at pH 7.5 containing 150 mM arginine in which 20 mg / mL of PEG-FGF21 maintains all quality attributes related to dimerization, aggregation, and degradation.

[0290] Formulations with and without PS80 were monitored for long-term stability over a period of 12 months. Figure 17-Figure 20 The long-term stability demonstrated that by controlling acid variants, dimers, microparticles, etc., PEG-FGF21 maintained all quality attributes while maintaining activity, thereby demonstrating suitability for storage as a liquid formulation.

[0291] Table 2

[0292]

[0293] Table 3 Test methods for stability study

[0294]

[0295]

[0296] Table 4 Appearance Results - 12 Month Stability

[0297]

[0298]

[0299]

[0300] Table 5 pH results - 12 month stability

[0301]

[0302]

[0303] Table 6 Protein concentration results - 12 month stability

[0304]

[0305] Table 7 Osmotic Molar Concentration Results T0 Data

[0306] Preparation code mOsm / kg 1 317 2 302 3 303

[0307] Table 8 SEC-HPLC results - 12 month stability

[0308]

[0309] Table 9 icIEF results - 12 month stability

[0310]

[0311] Table 10 RP-HPLC results - 12 month stability

[0312]

[0313] Table 11 MFI results - 12 month stability

[0314]

[0315] The stability data for Formulation 2 are shown in Tables 12 and 13.

[0316] Table 12: Long-term stability data of liquid formulations at 5±3°C

[0317]

[0318] NT = not tested or not planned; ND = not detected. HOA = higher level aggregates.

[0319] a = add test

[0320] Table 13: Accelerated stability data of liquid formulations at 25±2°C

[0321]

[0322]

[0323] NT = not tested or not planned; ND = not detected. HOA = higher level aggregates.

[0324] Table 14 shows the acceleration curves for a formulation comprising 28 mg / mL of mutant FGF21 peptide conjugate, 260 mM arginine HCl, 20 mM Tris, 0.2 mg / ml PS80, pH 7.1.

[0325] Table 14

[0326]

[0327] Table 15 below provides a ranking for further optimization based on additional excipient screening.

[0328] Table 15

[0329]

[0330]

[0331]

[0332] All disclosures mentioned herein are incorporated herein by reference in their entirety. Although the above invention has been described in detail for the purpose of clarity and understanding, those skilled in the art will appreciate from reading the disclosure that various changes in form and detail may be made without departing from the true scope of the invention in the appended claims.

[0333] For the purpose of illustration, specific embodiments of methods and kits have been described herein. These are merely embodiments. The technology provided herein can be applied to systems other than the example systems described above. Many changes, modifications, additions, omissions, and substitutions are possible in the practice of the present invention. The present invention includes changes to the described embodiments, which will be apparent to the skilled person, including changes obtained by: replacing features, elements, and / or actions with equivalent features, elements, and / or actions; mixing and matching features, elements, and / or actions from different embodiments; combining features, elements, and / or actions from the embodiments described herein with features, elements, and / or actions of other technologies; and / or omitting to combine features, elements, and / or actions from the described embodiments.

[0334] The embodiments of the present invention described above are intended to be exemplary only. Those skilled in the art will appreciate that various modifications of detail may be made to these embodiments, all of which are within the scope of the present invention.

Claims

1. A prefilled syringe or autoinjector comprising a liquid pharmaceutical composition, the liquid pharmaceutical composition comprising: (a) 28 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG; (b) 260 mM arginine; (c) 0.02% polysorbate 80 (PS-80); (d) 20 mM buffer, wherein the pH is 7.1; and (e) a pharmaceutically acceptable carrier.

2. A prefilled syringe or autoinjector comprising a liquid pharmaceutical composition comprising: (a) 36 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and the first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between the second position of the glycosyl moiety and the 20 kDa PEG. PEG; (b) 270 mM arginine; (c) 0.02% polysorbate 80 (PS-80); (d) 20 mM buffer, wherein the pH is 7.1; and (e) a pharmaceutically acceptable carrier.

3. A prefilled syringe or autoinjector comprising a liquid pharmaceutical composition comprising: (a) 42 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG; (b) 270 mM arginine; (c) 0.02% polysorbate 80 (PS-80); (d) 20 mM buffer, wherein the pH is 7.1; and (e) a pharmaceutically acceptable carrier.

4. The prefilled syringe or autoinjector according to any one of claims 1 to 3, wherein the arginine is arginine HCl or arginine sulfate.

5. A prefilled syringe or autoinjector comprising a liquid pharmaceutical composition comprising: (a) 28.8 to 43.2 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, the mutant FGF-21 peptide conjugate comprising: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG; (b) 160 to 240 mM arginine HCl; (c) 0.016% to 0.024% (w / v) polysorbate 80 (PS-80); (d) 16 to 24 mM Tris buffer, wherein the pH is 7 to 7.5; and (e) a pharmaceutically acceptable carrier.

6. The prefilled syringe or autoinjector according to any one of claims 1 to 5, wherein the liquid drug composition further comprises a surfactant.

7. The prefilled syringe or autoinjector of claim 6, wherein the surfactant comprises cetyltrimethylammonium bromide, sodium gluconate, or a combination thereof.

8. The prefilled syringe or autoinjector according to any one of claims 1-7, wherein the liquid drug composition comprises 0.05% to 0.1% (w / v) cetyltrimethylammonium bromide, 0.05% to 0.1% (w / v) sodium gluconate, or a combination thereof.

9. The prefilled syringe or autoinjector according to any one of claims 1 to 8, wherein the liquid drug composition is stable at a temperature in the range of 2°C to 8°C for up to 12 months.

10. The prefilled syringe or autoinjector according to any one of claims 1 to 9, wherein the liquid drug composition is stable at room temperature for at least 3 months.

11. The prefilled syringe or autoinjector according to any one of claims 4-10, wherein the molar ratio of the mutant FGF-21 peptide conjugate to the arginine HCl is 0.006 to 0.

009.

12. The prefilled syringe or autoinjector according to any one of claims 4-11, wherein the weight ratio of the mutant FGF-21 peptide conjugate to the arginine HCl is 0.6 to 0.

9.

13. The prefilled syringe or autoinjector according to any one of claims 1-11, wherein the liquid drug composition has an osmolality of 250 to 550 mOsmol / kg.

14. A liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG, The liquid pharmaceutical composition comprises 28 mg / ml of the mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, 260 mM arginine, 0.02% polysorbate 80 (PS-80), 20 mM buffer, and the pH is 7.

1. The liquid pharmaceutical composition is stable at a temperature of 2°C to 8°C for 12 months or longer.

15. A liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG, The liquid pharmaceutical composition comprises 36 mg / ml of the mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, 270 mM arginine, 0.02% polysorbate 80 (PS-80), 20 mM buffer, and the pH is 7.

1. The liquid pharmaceutical composition is stable at a temperature of 2°C to 8°C for 12 months or longer.

16. A liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG, The liquid pharmaceutical composition comprises 42 mg / ml of the mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, 270 mM arginine, 0.02% polysorbate 80 (PS-80), 20 mM buffer, and the pH is 7.

1. The liquid pharmaceutical composition is stable at a temperature of 2°C to 8°C for 12 months or longer.

17. A liquid pharmaceutical composition comprising a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, wherein the mutant FGF-21 peptide conjugate comprises: i) a mutant FGF-21 peptide comprising the amino acid sequence of SEQ ID NO: 2, ii) a glycosyl moiety, and iii) 20 kDa polyethylene glycol (PEG), wherein the mutant FGF-21 peptide is attached to the glycosyl moiety via a covalent bond between the threonine at amino acid position 173 of SEQ ID NO:2 and a first position of the glycosyl moiety, and wherein the glycosyl moiety is attached to the 20 kDa PEG via a covalent bond between a second position of the glycosyl moiety and the 20 kDa PEG, wherein the liquid pharmaceutical composition comprises 28.8 to 43.2 mg / ml of a mutant fibroblast growth factor-21 (FGF-21) peptide conjugate, 160 to 240 mM arginine HCl, 16 to 24 mM Tris, 0.016% to 0.024% (w / v) PS-80, and wherein the pH is 7 to 7.5, and The liquid pharmaceutical composition is stable at a temperature of 2°C to 8°C for 12 months or longer.

18. The liquid pharmaceutical composition according to any one of claims 14 to 16, wherein the arginine is arginine HCl or arginine sulfate.

19. The liquid pharmaceutical composition according to any one of claims 14 to 18, further comprising a surfactant.

20. The liquid pharmaceutical composition of claim 19, wherein the surfactant comprises cetyltrimethylammonium bromide, sodium gluconate, or a combination thereof.

21. The liquid pharmaceutical composition according to any one of claims 14 to 18, further comprising 0.05% to 0.1% (w / v) of cetyltrimethylammonium bromide, 0.05% to 0.1% (w / v) of sodium gluconate, or a combination thereof.

22. The liquid pharmaceutical composition according to any one of claims 14 to 21, wherein the liquid pharmaceutical composition is stable at room temperature for at least 3 months.

23. The liquid pharmaceutical composition according to any one of claims 17 to 22, wherein the weight ratio of the mutant FGF-21 peptide conjugate to the arginine HCl is 0.6 to 0.

9.

24. The liquid pharmaceutical composition according to any one of claims 17 to 23, wherein the molar ratio of the mutant FGF-21 peptide conjugate to the arginine HCl is 0.006 to 0.

009.

25. The liquid pharmaceutical composition according to any one of claims 14 to 24, wherein the liquid pharmaceutical composition has an osmotic concentration of 250 mOsmol / kg to 550 mOsmol / kg.

26. A container comprising the liquid pharmaceutical composition of any one of claims 14 to 25.

27. The container of claim 26, wherein the container is a prefilled syringe, a vial or an autoinjector.

28. A kit comprising the container of claim 26 or 27 and a label or instructions for administering and using the liquid pharmaceutical composition.

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