Pharmaceutical composition of human insulin analogue fusion protein and medical application thereof

Abstract

The invention relates to a pharmaceutical composition containing human insulin analogue fusion protein and application thereof. The pharmaceutical composition has good biological activity and stability.

Description

A pharmaceutical composition of human insulin analog fusion protein and its medical use Technical Field

[0001] The present disclosure relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition comprising a human insulin analog fusion protein, and pharmaceutical uses thereof. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by dysregulation of glucose, protein, and lipid metabolism in the human body due to insufficient insulin secretion. It is mainly divided into insulin-dependent diabetes mellitus (Type I diabetes) and non-insulin-dependent diabetes mellitus (Type II diabetes). 90-95% of diabetic patients worldwide suffer from Type II diabetes, which is caused by impaired pancreatic beta cell function and long-term insulin resistance. Its most prominent characteristics are a lack of insulin levels in the body, decreased insulin sensitivity, and high blood glucose concentrations in the plasma. Studies have shown that Type II diabetes is associated with a variety of high-risk complications, leading to cardiovascular disease, kidney failure, blindness, amputation, and many other complications.

[0003] Approved diabetes medications primarily include chemically synthesized small-molecule oral glucose-lowering drugs, such as biguanides, sulfonyl insulins, insulin sensitizers, α-glycosides, and injectable peptide glucose-lowering drugs like glucagon-like peptide-1 (GLP-1) analogs. For all patients with type 1 diabetes and some patients with advanced type 2 diabetes, only insulin injections can effectively control blood sugar levels.

[0004] Currently, approved insulin analogs are primarily categorized into two main categories: rapid-acting insulin and long-acting basal insulin. Approved rapid-acting insulins include insulin lispro and insulin aspart. Rapid-acting insulins are primarily administered subcutaneously before meals. By modifying the insulin molecule's structure, they inhibit the formation of native insulin hexamers, resulting in rapid onset and clearance after subcutaneous administration. Insulin lispro inhibits hexamer formation by alternating amino acids at positions 28 and 29 at the end of native insulin's B-chain, while insulin aspart replaces proline with aspartic acid at position 28 of the insulin B-chain, inhibiting hexamer formation through charge repulsion. Approved long-acting basal insulins include insulin glargine, insulin detemir, and insulin degludec. Insulin glargine introduces a positively charged arginine amino acid into the insulin backbone, raising its isoelectric point to neutral. This allows the insulin analog to precipitate at the site of administration after subcutaneous injection, resulting in significantly prolonged in vivo pharmacokinetic properties after sustained-release dissolution. Insulin detemir and insulin degludec are modified by introducing a long-chain fatty acid into the terminal lysine side chain of the B-chain, forming a supramolecular polymer at the administration site, and reversibly binding to human serum albumin in plasma to prolong their in vivo pharmacokinetic properties. Currently, insulin glargine and insulin degludec are administered once daily, while insulin detemir is administered once to twice daily.

[0005] However, fast-acting insulin products may cause adverse reactions such as hypoglycemia and severe hypoglycemia symptoms, and patients need to inject insulin drugs once or even multiple times a day, which makes it inconvenient for patients to use, has a heavy treatment burden, and reduces their quality of life. Existing long-acting basal insulin products also require subcutaneous injection once a day, and the injection frequency is still high. Therefore, ultra-long-acting basal insulin products with lower injection frequency and higher safety are the needs of clinical patients, which can significantly improve patient compliance and convenience. WO2023174370 provides such an ultra-long-acting basal insulin product.

[0006] However, fusion protein drugs have large molecular weights and complex structures, making them susceptible to degradation, polymerization, or undesirable chemical modifications, which can cause them to become unstable. In order to make fusion proteins suitable for drug administration and maintain stability during storage and subsequent use, thereby achieving better efficacy, research on stable formulations of fusion protein drugs is particularly important. For new insulin analog fusion proteins, it is still necessary to develop suitable pharmaceutical compositions.

[0007] Summary of the Invention

[0008] The present disclosure provides a pharmaceutical composition comprising a human insulin analog fusion protein, for example, a fusion protein comprising a human insulin analog and an IgG Fc region, wherein the composition has excellent stability.

[0009] The present disclosure provides a pharmaceutical composition comprising:

[0010] a) a fusion protein of a human insulin analog; and

[0011] b) a buffer, wherein the buffer is selected from one or more of histidine or a salt thereof, acetic acid or a salt thereof, succinic acid or a salt thereof, citrate and phosphate.

[0012] In some embodiments, the buffer is selected from one or more of histidine or a salt thereof, acetic acid or a salt thereof, and phosphate. In some embodiments, the buffer is selected from one or more of histidine or a salt thereof, and phosphate. In some embodiments, the buffer is selected from one or more of histidine, acetic acid, and disodium hydrogen phosphate. In some embodiments, the buffer is selected from one or more of histidine, acetate-histidine, and disodium hydrogen phosphate. In some embodiments, the buffer is selected from phosphate. In some embodiments, the buffer is acetate-histidine. In some embodiments, the buffer is selected from one or more of histidine and disodium hydrogen phosphate. In some embodiments, the buffer is histidine and disodium hydrogen phosphate. In some embodiments, the buffer is histidine.

[0013] In some embodiments, the aforementioned fusion protein comprises an insulin analog,

[0014] The insulin analog has the general formula: B1-L1-A1;

[0015] Among them, B1 is an insulin B chain analog, which comprises the following amino acid sequence:

[0016] FVX1QHLCGX2HLVEALX3X4VCGERGFX5X6X7X8X9 (SEQ ID NO: 43), wherein X1 is selected from N, G or K; X2 is selected from S or E; X3 is selected from Y, H or E; X4 is selected from H, R, L or E; X5 is selected from F or H; X6 is selected from G, T, S, H, V, Y or is absent; X7 is selected from G, E, P, K, D, S, H or is absent; X8 is selected from G, E, K, P, Q, D, H or is absent; X9 is selected from G, T, S, E, K, A or is absent;

[0017] A1 is an insulin A chain analogue comprising the following amino acid sequence:

[0018] GIVEQCCZ1SICSLZ2QLENYCZ3Z4 (SEQ ID NO: 44), wherein Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, and Z3 is selected from G or N; Z4 is any natural amino acid or does not exist. When Z3 is N, Z4 is not G or N.

[0019] L1 is a linker. In some embodiments, the aforementioned fusion protein comprises an insulin analog,

[0020] The insulin analog has the general formula: B1-L1-A1;

[0021] Among them, B1 is an insulin B chain analog, which comprises the following amino acid sequence:

[0022] FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1), wherein X1 is selected from N, G or K, X2 is selected from S or E, X3 is selected from Y, H or E, X4 is selected from H, R, L or E, and X5 is selected from F or H;

[0023] A1 is an insulin A chain analogue comprising the following amino acid sequence:

[0024] GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO: 2), wherein Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, and Z3 is selected from G or N;

[0025] L1 is a linker.

[0026] In some embodiments, B1 comprises (or is) the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 1, which has at least one, at least two, at least three, at least four, or five amino acid modifications at X1, X2, X3, X4, or X5. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, and / or X5 is selected from H. In some specific embodiments, X2 is E and X5 is H. In some specific embodiments, X5 is H. In some specific embodiments, X3 is H and X5 is H. In some specific embodiments, X1 is K, X2 is E, and X5 is H.

[0027] In some embodiments, A1 comprises (or is) the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 2, which has at least one, at least two, or three amino acid modifications at Z1, Z2, and Z3. In some specific embodiments, Z1 is selected from T, H, or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, Z2 is E. In some specific embodiments, Z1 is E and Z2 is E.

[0028] In some embodiments, B1 in the insulin analog comprises (or is) the sequence of SEQ ID NO:43 or SEQ ID NO:1, and A1 comprises (or is) the amino acid sequence of SEQ ID NO:44 or SEQ ID NO:2. In some specific embodiments, X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, and X5 is selected from H; and Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X5 is H, Z1 is H, Z2 is E, and Z3 is G. In some specific embodiments, X3 is H, X5 is H, and Z2 is E. In some specific embodiments, X1 is K, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, Z2 is E, and Z3 is G. In some specific embodiments, X2 is E, X5 is H, Z1 is E, and Z2 is E.

[0029] In some specific embodiments, X1 is N or K, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G;

[0030] X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is N;

[0031] X1 is N, X2 is S, X3 is Y, X4 is L, X5 is H, Z1 is H, Z2 is E, and Z3 is G;

[0032] X1 is N, X2 is S, X3 is H, X4 is L, X5 is H, Z1 is T, Z2 is E, and Z3 is G; or

[0033] X1 is N, X2 is E, X3 is Y, X4 is L, X5 is H, Z1 is E, Z2 is E, and Z3 is G.

[0034] In the above embodiments, X6-X9 or X7-X9 may each be G; or, X6-X9 or X7-X9 may not exist.

[0035] In the above embodiments, Z4 may be absent; or, when Z3 is N, Z4 is an amino acid other than G, N, S, V, L or P.

[0036] In some embodiments, B1 in the insulin analog comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 31-36:

[0037] In some embodiments, A1 in the insulin analog comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 37-42

[0038] In some embodiments, B1 and A1 in the insulin analog comprise (or are) the amino acid sequences shown in any one of the following groups: SEQ ID NOs: 31 and 37; SEQ ID NOs: 32 and 38; SEQ ID NOs: 33 and 39; SEQ ID NOs: 34 and 40; SEQ ID NOs: 35 and 41; SEQ ID NOs: 36 and 42.

[0039] In some embodiments, the linker shown in L1 is a polypeptide capable of achieving a connection function, for example, a flexible polypeptide. In some specific embodiments, the linker shown in L1 comprises (or is) the following sequences: including but not limited to G4S (SEQ ID NO: 53), GS, GAP, ASGS (SEQ ID NO: 54), polyguanine (poly G). In some specific embodiments, the linker shown in L1 comprises (or is) (G m S p ) n , wherein each m is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), each n is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), and each p is independently selected from 0 to 4. In some specific embodiments, the GS or poly G linker has at least 5 Gs.

[0040] In the present disclosure, GS linker encompasses any type of linker comprising amino acids G and S in the linker.

[0041] In some specific embodiments, the linker represented by L1 comprises (or is) GGSGGGG (SEQ ID NO:45), GSGGGG (SEQ ID NO:46), GGGGG (SEQ ID NO:47), GGGGGGSGGGG (SEQ ID NO:3) or GGGGGSGGGG (SEQ ID NO:52).

[0042] In some embodiments, any of the aforementioned fusion proteins further comprises a C1 domain, and the C1 domain can extend the half-life of the insulin analog.

[0043] In some embodiments, any of the aforementioned fusion proteins further comprises a C1 domain, wherein the C1 is selected from, for example, an Fc region of an immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM), an HSA protein or polypeptide, an HSA binding domain (e.g., an anti-HSA antibody or an antigen-binding fragment thereof), XTEN, a peptide glycine-rich high amino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer.

[0044] In some embodiments, the XTEN peptide is an unstructured, hydrophilic long peptide comprising varying percentages of the six amino acids Ala, Glu, Gly, Ser, and Thr, such as XTEN TM Peptide (Amunix Operating Inc.) In some embodiments, the PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala, and Ser residues and is at least about 100, 200, 300, 400, 500, or 600 amino acids in length.

[0045] In some embodiments, a fusion protein contains one or more (eg, two, three, four) insulin analogs and a C1.

[0046] In some embodiments, C1 is selected from the Fc region of human IgG1, IgG2, and IgG4. In some embodiments, the Fc region comprises a mutation selected from the group consisting of L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P or S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3, or IgG4, V234A / G237A on IgG2, K214T / E233P / L235A on IgG1, IgG2, IgG3, or IgG4, 8S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, for example, an Fc having residues 117-260 from IgG2 and residues 261-447 from IgG4. In some specific embodiments, the human IgG4 Fc region has S228P, F234A, L235A, and / or K447A mutations. In some specific embodiments, the human IgG1 Fc region has L234A / L235A or L234A / L235A / P329G mutations. In some embodiments, the mutation sites in the Fc region are numbered according to EU numbering conventions.

[0047] In some specific embodiments, C1 is selected from an amino acid sequence comprising (or as) any one of SEQ ID NOs: 17, 18, 48-51, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0048] >Human IgG1 Fc region 1

[0049] >Human IgG1 Fc region 2

[0050] >Human IgG4 Fc region 1

[0051] >Human IgG4 Fc region 2

[0052] In some embodiments, any of the aforementioned fusion proteins further comprises L2, wherein the linker represented by L2 is a polypeptide capable of achieving a connection function (e.g., a flexible polypeptide), and C1 forms a fusion protein with B1-L1-A1 through L2. In some specific embodiments, the linker represented by L2 comprises (or is) the following sequence: including but not limited to G4S, GS, GAP, ASGS, polyguanine (poly G), (G m Q) n 、(G m A) n 、(G m Q i ) n 、(G m A i ) n or (PGPQ) s (SEQ ID NO: 55), wherein each m is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), each n is independently selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6), each i is independently selected from an integer of 0-4 (e.g., 0, 1, 2, 3, 4), and s is selected from an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6). In some specific embodiments, L2 comprises (or is) the amino acid sequence of any one of SEQ ID NOs: 10-16.

[0053] In some embodiments, C1 in any of the aforementioned fusion proteins is located at the N-terminus of the insulin analog. In other embodiments, C1 in the fusion protein is located at the C-terminus of the insulin analog. In some specific embodiments, the fusion protein has the following sequence from N-terminus to C-terminus: B1-L1-A1-L2-C1, C1-L2-B1-L1-A1, A1-L1-B1-L2-C1, or C1-L2-A1-L1-B1.

[0054] In some embodiments, the insulin analog is an insulin receptor agonist having insulin receptor agonist activity.

[0055] In some embodiments, any of the aforementioned fusion proteins is a monomer, dimer, or multimer. In some specific embodiments, the dimer is a homodimer, wherein the amino acid sequences of the two fusion proteins constituting the dimer are identical or substantially identical. In other specific embodiments, the dimer is a heterodimer, wherein the amino acid sequences of the two fusion proteins constituting the dimer are different.

[0056] In some embodiments, any of the aforementioned fusion proteins comprises (or is) the amino acid sequence shown in any one of SEQ ID NOs: 19-27, or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0057] In some embodiments, any of the aforementioned fusion proteins comprises (or is) the amino acid sequence shown in SEQ ID NO:28.

[0058] In some embodiments, any of the aforementioned fusion proteins (e.g., any of SEQ ID NOs: 19-27, more preferably SEQ ID NOs: 19, 20, 21, or 27) has a reduced ability to promote cell proliferation, thereby having a reduced risk of inducing tumorigenesis and / or tumor development. The cells are, for example, cells expressing IGF-1R on their surface. In some embodiments, the reduction is the ability of the fusion protein to promote EC proliferation in cells (e.g., MCF-7) compared to natural human insulin. 50 The value is increased by 1 to 10 times, 1 to 100 times, 1 to 1000 times, 1 to 10000 times, 100 to 500 times, 200 to 400 times; for example, EC 50 The fusion protein 1 shown in SEQ ID NO: 19 has an ability to promote cell proliferation that is about 200 times lower than that of natural human insulin. The method of Example 5 of the present disclosure can be used to detect the EC of cells (e.g., MCF-7) that proliferate. 50 .

[0059] In some embodiments, any of the aforementioned fusion proteins (e.g., any of the sequences of SEQ ID NOs: 19-27, more e.g., SEQ ID NOs: 19 or 20) has a half-life of 1 to 10 days, 2 to 9 days, 2 to 8 days, or 3 to 7 days, such as about 1, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10 days. In some specific embodiments, the fusion protein 1 shown in SEQ ID NO: 19 has a half-life of about 4.5 days. The half-life of the protein can be detected using the method of Example 6 of the present disclosure.

[0060] In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 0.01 mg / mL - 500 mg / mL, such as 0.05 mg / mL - 450 mg / mL, 0.05 mg / mL - 400 mg / mL, 0.05 mg / mL - 350 mg / mL, 0.05 mg / mL - 300 mg / mL, 0.05 mg / mL - 250 mg / mL, 0.05 mg / mL - 200 mg / mL, 0.05 mg / mL - 150 mg / mL, 0.05 mg / mL - 140 mg / mL, 0.05 mg / mL - 130 mg / mL, 0.05 mg / mL - 120 mg / mL, 0.05 mg / mL - 110 mg / mL, 0.05 mg / mL - 100 mg / mL, 0.1 mg / mL - 400 mg / mL, 0.1 mg / mL - 350 mg / mL, 0.1 mg / mL - 300 mg / mL, 0.1 mg / mL - 250 mg / mL, 0.1 mg / mL - 200 mg / mL, 0.1 mg / mL - 150 mg / mL, 0.1 mg / mL - 140 mg / mL, 0.1 mg / mL - 130 mg / mL, 0.1 mg / mL - 120 mg / mL, 0.1 mg / mL - 110 mg / mL, 0.1 mg / mL - 100 mg / mL, 0.5 mg / mL - 350 mg / mL, 0.5 mg / mL - 300 mg / mL, 0.5 mg / mL - 250 mg / mL, 0.5 mg / mL - 200 mg / mL, 0.5 mg / mL - 150 mg / mL, 0.5 mg / mL - 140 mg / mL, 0.5 mg / mL - 130 mg / mL, 0.5 mg / mL - 120 mg / mL, 0.5 mg / mL - 110 mg / mL, 0.5 mg / mL - 100 mg / mL, 1 mg / mL - 300 mg / mL, 1 mg / mL - 250 mg / mL, 1 mg / mL - 200 mg / mL, 1 mg / mL - 150 mg / mL, 1 mg / mL - 140 mg / mL, 1 mg / mL - 130 mg / mL, 1 mg / mL - 120 mg / mL, 1 mg / mL - 110 mg / mL, 1 mg / mL - 100 mg / mL, 1 mg / mL - 95 mg / mL, 1 mg / mL - 90 mg / mL, 1 mg / mL - 85 mg / mL, 1 mg / mL - 80 mg / mL, 1 mg / mL - 75 mg / mL, 1 mg / mL - 70 mg / mL, 1 mg / mL - 65 mg / mL, 1 mg / mL - 60 mg / mL, 1 mg / mL - 55 mg / mL, 1 mg / mL - 50 mg / mL, 1 mg / mL - 45 mg / mL, 1 mg / mL - 40 mg / mL, 1 mg / mL - 35 mg / mL,1mg / mL-30mg / mL、1mg / mL-25mg / mL、1mg / mL-20mg / mL、1mg / mL-15mg / mL、5mg / mL-90mg / mL、5mg / mL-85mg / mL、5mg / mL-80mg / mL、5mg / mL-75mg / mL、5mg / mL-70mg / mL、5mg / mL-65mg / mL、5mg / mL-60mg / mL、5mg / mL-55mg / mL、5mg / mL-50mg / mL、5mg / mL-45mg / mL、5mg / mL-40mg / mL、5mg / mL-35mg / mL、5mg / mL-30mg / mL、5mg / mL-25mg / mL、 5mg / mL-20mg / mL、5mg / mL-15mg / mL、8mg / mL-12mg / mL、30mg / mL-250mg / mL、30mg / mL-200mg / mL、30mg / mL-190mg / mL、30mg / mL-180mg / mL、30mg / mL-170mg / mL、30mg / mL-160mg / mL、30mg / mL-150mg / mL、30mg / mL-140mg / mL、30mg / mL-130mg / mL、30mg / mL-120mg / mL、30mg / mL-110mg / mL、30mg / mL-100mg / mL、50mg / mL-200mg / mL、50mg / mL-190mg / mL、50mg / mL-180mg / mL、50mg / mL-170mg / mL、50mg / mL-160mg / mL、50mg / mL-150mg / mL、50mg / mL-140mg / mL、50mg / mL-130mg / mL、50mg / mL-120mg / mL、50mg / mL-110mg / mL、50mg / mL-100mg / mL、70mg / mL-180mg / mL、70mg / mL-170mg / mL、70mg / mL-160mg / mL、70mg / mL-150mg / mL、70mg / mL-140mg / mL、70mg / mL-130mg / mL、70mg / mL-120mg / mL、70mg / mL-110mg / mL、70mg / mL-100mg / mL、90mg / mL-110mg / mL、In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 95 mg / mL-105 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 0.1 mg / mL-400 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 0.5 mg / mL-200 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 1 mg / mL-150 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 5 mg / mL-45 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 50 mg / mL-110 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, or about 150 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is about 5 mg / mL, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, or about 60 mg / mL. In some embodiments, the concentration of the fusion protein in the pharmaceutical composition is about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL.

[0061] In some embodiments, the concentration of the buffer in the pharmaceutical composition is 1 mM-200 mM, for example, 5 mM-190 mM, 5 mM-180 mM, 5 mM-170 mM, 5 mM-160 mM, 5 mM-150 mM, 5 mM-140 mM, 5 mM-130 mM, 5 mM-120 mM, 5 mM-110 mM, 5 mM-100 mM, 5 mM-90 mM, 5 mM -80mM, 10mM-180mM, 10mM-170mM, 10mM-160mM, 10mM-150mM, 10mM-140mM, 10mM-130mM, 1 0mM-120mM, 10mM-110mM, 10mM-100mM, 10mM-90mM, 10mM-80mM, 20mM-170mM, 20mM-160mM, 20mM-150mM, 20mM-140mM, 20mM-130mM, 20mM-120mM, 20mM-110mM, 20mM-100mM, 2 0mM-90mM, 20mM-80mM, 40mM-160mM, 40mM-150mM, 40mM-140mM, 40mM-130mM, 40mM- In some embodiments, the concentration of the buffer in the pharmaceutical composition is 10mM-150mM or 5mM-150mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 40mM-120mM or 10mM-120mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is 60mM-100mM or 10mM-100mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. In some embodiments, the concentration of the buffer in the pharmaceutical composition is about 10 mM, about 40 mM, or about 80 mM.

[0062] In some embodiments, the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is selected from one or more of polysorbates and poloxamers. In some embodiments, the polysorbate is selected from one or more of polysorbate 20 and polysorbate 80. In some embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is a poloxamer, such as poloxamer 188.

[0063] In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.01 mg / mL-5 mg / mL, for example, 0.01 mg / mL-5 mg / mL, 0.01 mg / mL-4 mg / mL, 0.01 mg / mL-3 mg / mL, 0.01 mg / mL-2 mg / mL, 0.01 mg / mL-1 mg / mL, 0.01 mg / mL-0.5 mg / mL, 0.05 mg / mL-4 mg / mL, 0.0 5mg / mL-3mg / mL, 0.05mg / mL-2mg / mL, 0.05mg / mL-1mg / mL, 0.05mg / mL-0.5mg / mL, 0.08mg / mL-3mg / mL ,0.08mg / mL-2.5mg / mL, 0.08mg / mL-2mg / mL, 0.08mg / mL-1.5mg / mL, 0.08mg / mL-1mg / mL, 0.08mg / mL-0 .5mg / mL, 0.1mg / mL-2.5mg / mL, 0.1mg / mL-2mg / mL, 0.1mg / mL-1.5mg / mL, 0.1mg / mL-1mg / mL, 0.1mg / m L-0.5mg / mL, 0.2mg / mL-2.5mg / mL, 0.2mg / mL-2mg / mL, 0.2mg / mL-1.5mg / mL, 0.2mg / mL-1mg / mL, 0.2mg In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.05 mg / mL to 3 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.08 mg / mL to 1.5 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is 0.1 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, the concentration of the surfactant in the pharmaceutical composition is about 0.2 mg / mL or about 0.4 mg / mL.

[0064] In some embodiments, the pharmaceutical composition further comprises one or more of a carbohydrate and a polyol. In some embodiments, the polyol is selected from one or more of mannitol, sorbitol, and glycerol. In some embodiments, the carbohydrate is selected from one or more of sucrose and trehalose. In some embodiments, the carbohydrate is sucrose. In some embodiments, the polyol is sorbitol or glycerol. In some embodiments, the polyol is trehalose. In some embodiments, the polyol is mannitol.

[0065] In some embodiments, the concentration of saccharides or polyols in the pharmaceutical composition is 1 mg / mL - 200 mg / mL, such as 1 mg / mL - 180 mg / mL, 1 mg / mL - 170 mg / mL, 1 mg / mL - 160 mg / mL, 1 mg / mL - 150 mg / mL, 1 mg / mL - 140 mg / mL, 1 mg / mL - 130 mg / mL, 1 mg / mL - 120 mg / mL, 1 mg / mL - 110 mg / mL, 1 mg / mL - 100 mg / mL, 1 mg / mL - 90 mg / mL, 1 mg / mL - 80 mg / mL, 1 mg mg / mL - 70 mg / mL, 1 mg / mL - 60 mg / mL, 1 mg / mL - 50 mg / mL, 5 mg / mL - 160 mg / mL, 5 mg / mL - 150 mg / mL, 5 mg / mL - 140 mg / mL, 5 mg / mL - 130 mg / mL, 5 mg / mL - 120 mg / mL, 5 mg / mL - 110 mg / mL, 5 mg / mL - 100 mg / mL, 5 mg / mL - 90 mg / mL, 5 mg / mL - 80 mg / mL, 5 mg / mL - 70 mg / mL, 5 mg / mL - 60 mg / mL, 5 mg / mL - 50 mg / mL, 10 mg / mL - 150 mg / mL, 10 mg / mL - 140 mg / mL, 10 mg / mL - 130 mg / mL, 10 mg / mL - 120 mg / mL, 10 mg / mL - 110 mg / mL, 10 mg / mL - 100 mg / mL, 10 mg / mL - 90 mg / mL, 10 mg / mL - 80 mg / mL, 10 mg / mL - 70 mg / mL, 10 mg / mL - 60 mg / mL, 10 mg / mL - 50 mg / mL, 20 mg / mL - 140 mg / mL, 20 mg / mL - 130 mg / mL, 20 mg / mL - 120 mg / mL, 20 mg / mL - 110 mg / mL, 20 mg / mL - 100 mg / mL, 20 mg / mL - 90 mg / mL, 20 mg / mL - 80 mg / mL, 20 mg / mL - 70 mg / mL, 20 mg / mL - 60 mg / mL, 20 mg / mL - 50 mg / mL, 30 mg / mL - 130 mg / mL, 30 mg / mL - 120 mg / mL, 30 mg / mL - 110 mg / mL, 30 mg / mL - 100 mg / mL, 30 mg / mL - 90 mg / mL, 30 mg / mL - 80 mg / mL, 30 mg / mL - 70 mg / mL, 30 mg / mL - 60 mg / mL, 30 mg / mL - 50 mg / mL, 40 mg / mL - 120 mg / mL, 40 mg / mL - 110 mg / mL, 40 mg / mL - 100 mg / mL,In some embodiments, the concentration of the saccharide or polyol in the pharmaceutical composition is 10 mg / mL to 150 mg / mL. In some embodiments, the concentration of the carbohydrate or polyol in the pharmaceutical composition is 30 mg / mL to 120 mg / mL. In some embodiments, the concentration of the carbohydrate or polyol in the pharmaceutical composition is 40 mg / mL to 100 mg / mL.

[0066] In some embodiments, the concentration of sucrose or trehalose in the pharmaceutical composition is 1 mg / mL-150 mg / mL, for example, 5 mg / mL-140 mg / mL, 5 mg / mL-130 mg / mL, 5 mg / mL-120 mg / mL, 5 mg / mL-110 mg / mL, 5 mg / mL-100 mg / mL, 5 mg / mL-90 mg / mL, 5 mg / mL-80 mg / mL, 5 mg / mL-70 mg / mL, 10 mg / mL-130 mg / mL, 10 mg / mL-120 mg / mL. L, 10mg / mL-110mg / mL, 10mg / mL-100mg / mL, 10mg / mL-90mg / mL, 10mg / mL-80mg / mL, 10mg / mL-70mg / mL, 20mg / mL-130mg / mL , 20mg / mL-120mg / mL, 20mg / mL-110mg / mL, 20mg / mL-100mg / mL, 20mg / mL-90mg / mL, 20mg / mL-80mg / mL, 20mg / mL-70mg / mL,30mg / mL-130mg / mL, 30mg / mL-110mg / mL, 30mg / mL-100mg / mL, 30mg / mL-90mg / mL, 30mg / mL-80mg / mL, 30m g / mL-70mg / mL, 40mg / mL-120mg / mL, 40mg / mL-110mg / mL, 40mg / mL-100mg / mL, 40mg / mL-90mg / mL, 40mg / mL -80mg / mL, 40mg / mL-70mg / mL, 50mg / mL-120mg / mL, 50mg / mL-110mg / mL, 50mg / mL-100mg / mL, 50mg / mL-100 mg / mL, 50mg / mL-90mg / mL, 50mg / mL-80mg / mL, 50mg / mL-70mg / mL, 60mg / mL-120mg / mL, 60mg / mL-110mg / mL , 60mg / mL-100mg / mL, 60mg / mL-90mg / mL, 60mg / mL-80mg / mL, 60mg / mL-70mg / mL, 65mg / mL-110mg / mL, 65m g / mL-100mg / mL, 65mg / mL-90mg / mL, 65mg / mL-80mg / mL, 65mg / mL-70mg / mL, 70mg / mL-110mg / mL, 70mg / mL- In some embodiments, the sucrose concentration in the pharmaceutical composition is 100 mg / mL, 70 mg / mL-95 mg / mL, 70 mg / mL-90 mg / mL, 75 mg / mL-110 mg / mL, 75 mg / mL-100 mg / mL, 75 mg / mL-95 mg / mL, 75 mg / mL-90 mg / mL, 80 mg / mL-110 mg / mL, 80 mg / mL-100 mg / mL, 80 mg / mL-95 mg / mL or 80 mg / mL-90 mg / mL. In some embodiments, the sucrose concentration in the pharmaceutical composition is 10 mg / mL-120 mg / mL. In some embodiments, the sucrose concentration in the pharmaceutical composition is 20 mg / mL-100 mg / mL. In some embodiments, the sucrose concentration in the pharmaceutical composition is 50 mg / mL-90 mg / mL. In some embodiments, the sucrose concentration in the pharmaceutical composition is about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, or about 90 mg / mL. In some embodiments, the sucrose concentration in the pharmaceutical composition is about 60 mg / mL, about 70 mg / mL, or about 80 mg / mL.

[0067] In some embodiments, the concentration of trehalose in the pharmaceutical composition is 5 mg / mL to 140 mg / mL. In some embodiments, the concentration of trehalose in the pharmaceutical composition is 30 mg / mL to 130 mg / mL. In some embodiments, the concentration of trehalose in the pharmaceutical composition is 60 mg / mL to 120 mg / mL. In some embodiments, the concentration of trehalose in the pharmaceutical composition is 75 mg / mL to 100 mg / mL. In some embodiments, the concentration of trehalose in the pharmaceutical composition is about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, or about 100 mg / mL. In some embodiments, the concentration of trehalose in the pharmaceutical composition is about 88 mg / mL.

[0068] In some embodiments, the concentration of sorbitol or mannitol in the pharmaceutical composition is 1 mg / mL-100 mg / mL, for example, 5 mg / mL-100 mg / mL, 5 mg / mL-90 mg / mL, 5 mg / mL-80 mg / mL, 5 mg / mL-70 mg / mL, 5 mg / mL-60 mg / mL, 5 mg / mL-50 mg / mL, 10 mg / mL-90 mg / mL, 10 mg / mL-80 mg / mL, 10 mg / mL-70 mg / mL, 10 mg / mL-60 mg / mL, 10 mg / mL-50 mg / mL, 20 mg / mL-70 mg / mL. / mL, 20mg / mL-65mg / mL, 20mg / mL-60mg / mL, 20mg / mL-55mg / mL, 20mg / mL-50mg / mL, 30mg / mL-70mg / mL, 30mg / mL-65mg / mL, 30mg / mL-60mg / m L, 30mg / mL-55mg / mL, 30mg / mL-50mg / mL, 35mg / mL-70mg / mL, 35mg / mL-65mg / mL, 35mg / mL-60mg / mL, 35mg / mL-55mg / mL, 35mg / mL-50mg / mL, 40mg / mL-60mg / mL, 40mg / mL-55mg / mL or 40mg / mL-50mg / mL.

[0069] In some embodiments, the concentration of sorbitol in the pharmaceutical composition is 10 mg / mL to 80 mg / mL. In some embodiments, the concentration of sorbitol in the pharmaceutical composition is 20 mg / mL to 70 mg / mL. In some embodiments, the concentration of sorbitol in the pharmaceutical composition is 30 mg / mL to 60 mg / mL. In some embodiments, the concentration of sorbitol in the pharmaceutical composition is about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, or about 60 mg / mL. In some embodiments, the concentration of sorbitol in the pharmaceutical composition is about 45 mg / mL.

[0070] In some embodiments, the concentration of mannitol in the pharmaceutical composition is 10mg / mL-80mg / mL. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 20mg / mL-70mg / mL. In some embodiments, the concentration of mannitol in the pharmaceutical composition is 30mg / mL-60mg / mL. In some embodiments, the concentration of mannitol in the pharmaceutical composition is about 40mg / mL, about 41mg / mL, about 42mg / mL, about 43mg / mL, about 44mg / mL, about 45mg / mL, about 46mg / mL, about 47mg / mL, about 48mg / mL, about 49mg / mL, about 50mg / mL, about 51mg / mL, about 52mg / mL, about 53mg / mL, about 54mg / mL, about 55mg / mL, about 56mg / mL, about 57mg / mL, about 58mg / mL, about 59mg / mL or about 60mg / mL. In some embodiments, the concentration of mannitol in the pharmaceutical composition is about 50 mg / mL.

[0071] In some embodiments, the pharmaceutical composition further comprises a bacteriostatic agent comprising or selected from one or more of benzyl alcohol, phenol, and m-cresol. In some embodiments, the bacteriostatic agent comprises or is benzyl alcohol. In some embodiments, the bacteriostatic agent comprises or is phenol. In some embodiments, the bacteriostatic agent comprises or is m-cresol.

[0072] In some embodiments, the concentration of the antibacterial agent in the pharmaceutical composition is 0.01 mg / mL-50 mg / mL, for example, 0.05 mg / mL-45 mg / mL, 0.05 mg / mL-40 mg / mL, 0.05 mg / mL-35 mg / mL, 0.05 mg / mL-30 mg / mL, 0.05 mg / mL-25 mg / mL, 0.05 mg / mL-20 mg / mL, 0.05 mg / mL-15 mg / mL , 0.05mg / mL-10mg / mL, 0.05mg / mL-5mg / mL, 0.1mg / mL-40mg / mL, 0.1mg / mL-35mg / mL, 0.1mg / mL-30mg / mL, 0.1mg / mL-25mg / mL, 0.1mg / mL-20mg / mL, 0.1mg / mL-15mg / mL, 0.1mg / mL-10mg / mL, 0.1mg / mL-5m g / mL, 0.5mg / mL-35mg / mL, 0.5mg / mL-30mg / mL, 0.5mg / mL-25mg / mL, 0.5mg / mL-20mg / mL, 0.5mg / mL-1 5mg / mL, 0.5mg / mL-10mg / mL, 0.5mg / mL-5mg / mL, 1mg / mL-30mg / mL, 1mg / mL-25mg / mL, 1mg / mL-20mg / m L, 1mg / mL-15mg / mL, 1mg / mL-10mg / mL, 1mg / mL-5mg / mL, 2mg / mL-25mg / mL, 2mg / mL-20mg / mL, 2mg / mL-15mg / mL, 2mg / mL-10mg / mL, 2mg / mL-5mg / mL, 5mg / mL-20mg / mL, 5mg / mL-15mg / mL or 5mg / mL-10mg / mL. In some embodiments, the concentration of the bacteriostatic agent in the pharmaceutical composition is 0.05mg / mL-40mg / mL. In some embodiments, the concentration of the bacteriostatic agent in the pharmaceutical composition is 0.1mg / mL-30mg / mL. In some embodiments, the concentration of the bacteriostatic agent in the pharmaceutical composition is 0.5mg / mL-20mg / mL.

[0073] In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is 0.05 mg / mL-40 mg / mL, for example, 0.05 mg / mL-35 mg / mL, 0.05 mg / mL-30 mg / mL, 0.05 mg / mL-25 mg / mL, 0.05mg / mL-20mg / mL, 0.05mg / mL-15mg / mL, 0.05mg / mL-10mg / mL, 0.05mg / mL-5mg / mL, 0.1mg / mL-40mg / mL, 0.1mg / mL-35mg / mL, 0.1mg / mL-30mg / mL , 0.1mg / mL-25mg / mL, 0.1mg / mL-20mg / mL, 0.1mg / mL-15mg / mL, 0.1mg / mL-10mg / mL, 0.5mg / mL-35mg / mL, 0.5mg / mL-30mg / mL, 0.5mg / mL-25mg / mL, 0. In some embodiments, the benzyl alcohol concentration in the pharmaceutical composition is 0.1 mg / mL to 30 mg / mL. In some embodiments, the benzyl alcohol concentration in the pharmaceutical composition is 0.5 mg / mL to 20 mg / mL. In some embodiments, the benzyl alcohol concentration in the pharmaceutical composition is 0.5 mg / mL to 20 mg / mL. In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is 1 mg / mL to 15 mg / mL. In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, or about 15 mg / mL. In some embodiments, the concentration of benzyl alcohol in the pharmaceutical composition is about 5 mg / mL or about 10 mg / mL.

[0074] In some embodiments, the concentration of phenol in the pharmaceutical composition is 0.05 mg / mL-40 mg / mL, for example, 0.05 mg / mL-35 mg / mL, 0.05 mg / mL-30 mg / mL, 0.05 mg / mL-25 mg / mL, 0.05 mg / mL-20 mg / mL, 0.05 mg / mL-15 mg / mL, 0.05 mg / mL-10 mg / mL, 0.05 mg / mL-5 mg / mL, 0.1 mg / mL-30 mg / mL, 0.1 mg / mL-25 mg / mL, 0.1 mg / mL-20 mg / mL, 0.1 mg / mL-15 In some embodiments, the phenol concentration in the pharmaceutical composition is 0.1 mg / mL to 30 mg / mL. In some embodiments, the phenol concentration in the pharmaceutical composition is 0.5 mg / mL to 15 mg / mL. In some embodiments, the phenol concentration in the pharmaceutical composition is 1 mg / mL to 10 mg / mL. In some embodiments, the concentration of phenol in the pharmaceutical composition is about 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, or 10 mg / mL. In some embodiments, the concentration of phenol in the pharmaceutical composition is about 2.5 mg / mL or 5 mg / mL.

[0075] In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of 0.01 mg / mL-30 mg / mL, for example, 0.01 mg / mL-30 mg / mL, 0.01 mg / mL-25 mg / mL, 0.01 mg / mL-20 mg / mL, 0.01 mg / mL-15 mg / mL, 0.01 mg / mL-10 mg / mL, 0.01 mg / mL-5 mg / mL, 0.05 mg / mL-25 mg / mL, 0.05 mg / mL-20 mg / mL, 0.05 mg / mL-15 mg / mL, 0.05 mg / mL-10 mg / mL, 0.05 mg / mL-5 mg / mL, 0.1 mg / mL-20 mg / mL, 0.1 mg / mL-15 mg / mL, 0.1 mg / mL-10 mg / mL, 0.1 mg / mL-5 mg / mL, 0.5 mg / mL-15 mg / mL, In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of 0.5 mg / mL to 12 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 8 mg / mL, 0.5 mg / mL to 5 mg / mL, 1 mg / mL to 10 mg / mL, 1 mg / mL to 8 mg / mL, or 1 mg / mL to 5 mg / mL. In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of 0.05 mg / mL to 15 mg / mL. In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of 0.1 mg / mL to 10 mg / mL. In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of 0.5 mg / mL to 5 mg / mL. In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of about 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL. In some embodiments, the pharmaceutical composition has an intermediate cresol concentration of about 1 mg / mL or 3 mg / mL. In some embodiments, when the antibacterial agent in the pharmaceutical composition is benzyl alcohol (e.g., 1 mg / mL-15 mg / mL, e.g., 5 mg / mL-10 mg / mL), the composition further comprises a surfactant (e.g., 0.1 mg / mL-1 mg / mL, e.g., 0.2 mg / mL-0.4 mg / mL), and the surfactant is a polysorbate (e.g., polysorbate 80). In some embodiments, when the antibacterial agent in the pharmaceutical composition is phenol (e.g., 1 mg / mL-10 mg / mL, e.g., 2.5 mg / mL-5 mg / mL, e.g., about 2.5 mg / mL or about 5 mg / mL), the composition further comprises a surfactant (e.g., 0.4 mg / mL-2.5 mg / mL, e.g., 0.4 mg / mL-1 mg / mL, e.g., about 0.4 mg / mL), and the surfactant is a polysorbate, e.g., polysorbate 80.

[0076] In some embodiments, the pH of the buffer in the pharmaceutical composition is 5-9.5, e.g., 5.5-9, 5.5-8.5, 5.5-8, 5.5-7.5, 5.5-7, 6-9, 6-8.5, 6-8, 6-7.5, 6-7, 6.5-8.5, 6.5-8, 6.5-7.5, 6.5-7, 7-8, or 7-7.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 6-9. In some embodiments, the pH of the buffer in the pharmaceutical composition is 6.5-8.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is 7-8. In some embodiments, the pH of the buffer in the pharmaceutical composition is 6.5-7.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5. In some embodiments, the pH of the buffer in the pharmaceutical composition is about 6.5, about 7.0, or about 7.5.

[0077] In some embodiments, the pharmaceutical composition further comprises a pH adjusting agent, such as sodium hydroxide and / or hydrochloric acid.

[0078] In some embodiments, the pH of the pharmaceutical composition differs by no more than ±0.5 from the pH of the buffer it contains. In some embodiments, the pH of the pharmaceutical composition is 5-9.5, such as 5.5-9, 5.5-8.5, 5.5-8, 5.5-7.5, 5.5-7, 6-9, 6-8.5, 6-8, 6-7.5, 6-7, 6.5-8.5, 6.5-8, 6.5-7.5, 6.5-7, 7-8, or 7-7.5. In some embodiments, the pH of the pharmaceutical composition is 6-9. In some embodiments, the pH of the pharmaceutical composition is 6.5-8.5. In some embodiments, the pH of the pharmaceutical combination is 7-8. In some embodiments, the pH of the buffer in the pharmaceutical composition is 6.5-7.5. In some embodiments, the pH of the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0. In some embodiments, the pH of the pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5. In some embodiments, the pH of the pharmaceutical composition is about 6.5, about 7.0, or about 7.5.

[0079] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a fusion protein of a human insulin analog (e.g., as shown in the amino acid sequence of SEQ ID NO: 19), comprising any one of the following groups 1) to 8):

[0080] 1) Fusion protein;

[0081] A buffer selected from one or more of histidine or its salts and phosphates, for example, one or more of histidine, acetate-histidine and disodium hydrogen phosphate, and another example is histidine;

[0082] 2) fusion protein;

[0083] A buffer selected from one or more of histidine or its salts and phosphates, for example, one or more of histidine, acetate-histidine and disodium hydrogen phosphate, and another example is histidine;

[0084] polysorbates or poloxamers;

[0085] sucrose, sorbitol, or glycerin;

[0086] Optionally, the composition further comprises a bacteriostatic agent, for example comprising or selected from one or more of benzyl alcohol, phenol and m-cresol;

[0087] 3) fusion protein;

[0088] a buffering agent, which is histidine;

[0089] Polysorbate;

[0090] mannitol or trehalose;

[0091] Optionally, the composition further comprises a bacteriostatic agent, for example comprising or selected from one or more of benzyl alcohol, phenol and m-cresol;

[0092] 4) fusion proteins;

[0093] A buffer selected from one or more of histidine or its salts and phosphates, for example, one or more of histidine, acetate-histidine and disodium hydrogen phosphate, and another example is histidine;

[0094] and the pH of the pharmaceutical composition is 7-8, for example, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0;

[0095] 5) fusion protein;

[0096] A buffer selected from one or more of histidine or its salts and phosphates, for example, one or more of histidine, acetate-histidine and disodium hydrogen phosphate, and another example is histidine;

[0097] and the pH of the pharmaceutical composition is 6.5-7.5, for example, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5;

[0098] 6) fusion proteins;

[0099] A buffer, for example, one or more selected from histidine or its salts and phosphates, for example, one or more selected from histidine, acetate-histidine and disodium hydrogen phosphate, and another example is histidine;

[0100] bacteriostatic agent, wherein, i) when the bacteriostatic agent comprises or is benzyl alcohol (e.g., 1 mg / mL-15 mg / mL, e.g., 5 mg / mL-10 mg / mL), the composition further comprises a surfactant (e.g., 0.1 mg / mL-1 mg / mL, e.g., 0.2 mg / mL-0.4 mg / mL), wherein the surfactant is a polysorbate (e.g., polysorbate 80) or a poloxamer (e.g., poloxamer 188); or,

[0101] ii) when the bacteriostatic agent comprises or is phenol (e.g., 1 mg / mL-10 mg / mL, such as 2.5 mg / mL-5 mg / mL, such as about 2.5 mg / mL or about 5 mg / mL), the composition further comprises a surfactant (e.g., 0.4 mg / mL-2.5 mg / mL, such as 0.4 mg / mL-1 mg / mL, such as about 0.4 mg / mL), and the surfactant is a polysorbate or a poloxamer, such as polysorbate 80 or a poloxamer;

[0102] 7) fusion proteins;

[0103] a buffer selected from one or more of histidine, acetate-histidine and disodium hydrogen phosphate, for example, histidine;

[0104] bacteriostatic agent, wherein, i) when the bacteriostatic agent comprises or is benzyl alcohol (e.g., 1 mg / mL-15 mg / mL, e.g., 5 mg / mL-10 mg / mL), the composition further comprises a surfactant (e.g., 0.1 mg / mL-1 mg / mL, e.g., 0.2 mg / mL-0.4 mg / mL), wherein the surfactant is a polysorbate (e.g., polysorbate 80) or a poloxamer (e.g., poloxamer 188); or,

[0105] ii) when the bacteriostatic agent comprises or is phenol (e.g., 1 mg / mL-10 mg / mL, such as 2.5 mg / mL-5 mg / mL, such as about 2.5 mg / mL or about 5 mg / mL), the composition further comprises a surfactant (e.g., 0.4 mg / mL-2.5 mg / mL, such as 0.4 mg / mL-1 mg / mL, such as about 0.4 mg / mL), and the surfactant is a polysorbate or poloxamer, such as polysorbate 80;

[0106] The pH of the pharmaceutical composition is 7-8, for example, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0107] 8) fusion proteins;

[0108] a buffer selected from one or more of histidine, acetate-histidine and disodium hydrogen phosphate, for example, histidine;

[0109] bacteriostatic agent, wherein, i) when the bacteriostatic agent comprises or is benzyl alcohol (e.g., 1 mg / mL-15 mg / mL, e.g., 5 mg / mL-10 mg / mL), the composition further comprises a surfactant (e.g., 0.1 mg / mL-1 mg / mL, e.g., 0.2 mg / mL-0.4 mg / mL), wherein the surfactant is a polysorbate (e.g., polysorbate 80) or a poloxamer (e.g., poloxamer 188); or,

[0110] ii) when the bacteriostatic agent comprises or is phenol (e.g., 1 mg / mL-10 mg / mL, such as 2.5 mg / mL-5 mg / mL, such as about 2.5 mg / mL or about 5 mg / mL), the composition further comprises a surfactant (e.g., 0.4 mg / mL-2.5 mg / mL, such as 0.4 mg / mL-1 mg / mL, such as about 0.4 mg / mL), and the surfactant is a polysorbate or poloxamer, such as polysorbate 80;

[0111] And the 6.5-7.5 of the pharmaceutical composition is, for example, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, and about 7.5.

[0112] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a fusion protein of a human insulin analog (e.g., as shown in the amino acid sequence of SEQ ID NO: 19), which comprises or is any one of the following groups 1) to 22):

[0113] 1) Fusion protein; histidine; polysorbate; sucrose and water for injection;

[0114] 2) Fusion protein; histidine; polysorbate; sucrose; benzyl alcohol and water for injection;

[0115] 3) Fusion protein; histidine; polysorbate; sucrose; phenol and water for injection;

[0116] 4) Fusion protein; histidine; polysorbate; sucrose; m-cresol and water for injection;

[0117] 5) Fusion protein; histidine; polysorbate; sorbitol and water for injection;

[0118] 6) Fusion protein; histidine; polysorbate; sorbitol; benzyl alcohol and water for injection;

[0119] 7) Fusion protein; histidine; polysorbate; sorbitol; phenol and water for injection;

[0120] 8) Fusion protein; histidine; polysorbate; sorbitol; m-cresol and water for injection;

[0121] 9) Fusion protein; histidine; polysorbate; mannitol and water for injection;

[0122] 10) Fusion protein; histidine; polysorbate; mannitol; benzyl alcohol and water for injection;

[0123] 11) Fusion protein; histidine; polysorbate; mannitol; phenol and water for injection;

[0124] 12) Fusion protein; histidine; polysorbate; mannitol; m-cresol and water for injection;

[0125] 13) Fusion protein; histidine; polysorbate; trehalose and water for injection;

[0126] 14) Fusion protein; histidine; polysorbate; trehalose; benzyl alcohol and water for injection;

[0127] 15) Fusion protein; histidine; polysorbate; trehalose; phenol and water for injection;

[0128] 16) Fusion protein; histidine; polysorbate; trehalose; m-cresol and water for injection;

[0129] 17) Fusion protein; histidine; poloxamer; sucrose and water for injection;

[0130] 18) Fusion protein; histidine; poloxamer; sucrose; benzyl alcohol and water for injection;

[0131] 19) Fusion protein; histidine; poloxamer; sucrose; phenol and water for injection;

[0132] 20) Fusion protein; histidine; poloxamer; sucrose; m-cresol and water for injection;

[0133] 21) is any one of the pharmaceutical compositions of 1) to 16), wherein the polysorbate is polysorbate 80;

[0134] 22) is the pharmaceutical composition of any one of 17) to 21), wherein the polysorbate is replaced by poloxamer 188.

[0135] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a fusion protein of a human insulin analog (e.g., as shown in the amino acid sequence of SEQ ID NO: 19), comprising any one of the following groups 1) to 12):

[0136] 1) 0.01 mg / mL-500 mg / mL fusion protein;

[0137] 1mM-200mM buffer selected from one or more of histidine or its salts, and phosphates, for example, one or more of histidine and disodium hydrogen phosphate;

[0138] 0.01mg / mL-5mg / mL polysorbate or poloxamer;

[0139] 1mg / mL-200mg / mL sucrose or sorbitol;

[0140] Optionally, the pharmaceutical composition further comprises 0.01 mg / mL-50 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0141] and the pH of the pharmaceutical composition is 5-9.5;

[0142] 2) 0.1 mg / mL-400 mg / mL fusion protein;

[0143] 5mM-150mM buffer selected from one or more of histidine or its salts, and phosphates, for example, one or more of histidine and disodium hydrogen phosphate;

[0144] 0.05mg / mL-3mg / mL polysorbate or poloxamer;

[0145] 10mg / mL-150mg / mL sucrose or sorbitol;

[0146] Optionally, the pharmaceutical composition further comprises 0.05 mg / mL-40 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0147] and the pH of the pharmaceutical composition is 6-9;

[0148] 3) 0.5 mg / mL-200 mg / mL fusion protein;

[0149] 10mM-120mM buffer selected from one or more of histidine or its salts, and phosphates, for example, one or more of histidine and disodium hydrogen phosphate;

[0150] 0.08mg / mL-1.5mg / mL polysorbate or poloxamer;

[0151] 30mg / mL-120mg / mL sucrose or sorbitol;

[0152] Optionally, the pharmaceutical composition further comprises 0.1 mg / mL-30 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0153] and the pH of the pharmaceutical composition is 6.5-8.5;

[0154] 4) 1 mg / mL-150 mg / mL fusion protein;

[0155] 10mM-100mM buffer selected from one or more of histidine or its salts, and phosphates, for example, one or more of histidine and disodium hydrogen phosphate;

[0156] 0.1mg / mL-1mg / mL polysorbate or poloxamer;

[0157] 40mg / mL-100mg / mL sucrose or sorbitol;

[0158] Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0159] and the pH of the pharmaceutical composition is 6.5-7.5;

[0160] 5) 0.01 mg / mL-500 mg / mL fusion protein;

[0161] 1mM-200mM buffer selected from one or more of histidine and disodium hydrogen phosphate, for example, histidine;

[0162] 0.01mg / mL-5mg / mL polysorbate;

[0163] 1mg / mL-200mg / mL sucrose or sorbitol;

[0164] Optionally, the pharmaceutical composition further comprises 0.01 mg / mL-50 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0165] and the pH of the pharmaceutical composition is 5-9.5;

[0166] 6) 0.1 mg / mL-400 mg / mL fusion protein;

[0167] 10mM-150mM buffer selected from one or more of histidine and disodium hydrogen phosphate, for example, histidine;

[0168] 0.05mg / mL-3mg / mL polysorbate;

[0169] 10mg / mL-150mg / mL sucrose or sorbitol;

[0170] Optionally, the pharmaceutical composition further comprises 0.05 mg / mL-40 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0171] and the pH of the pharmaceutical composition is 6-9;

[0172] 7) 0.5 mg / mL-200 mg / mL fusion protein;

[0173] 40mM-120mM buffer selected from one or more of histidine and disodium hydrogen phosphate, for example, histidine;

[0174] 0.08mg / mL-1.5mg / mL polysorbate;

[0175] 30mg / mL-120mg / mL sucrose or sorbitol;

[0176] Optionally, the pharmaceutical composition further comprises 0.1 mg / mL-30 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0177] and the pH of the pharmaceutical composition is 6.5-8.5;

[0178] 8) 1 mg / mL-150 mg / mL fusion protein;

[0179] 60mM-100mM buffer selected from one or more of histidine and disodium hydrogen phosphate, for example, histidine;

[0180] 0.1mg / mL-1mg / mL polysorbate;

[0181] 40mg / mL-100mg / mL sucrose or sorbitol;

[0182] Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0183] and the pH of the pharmaceutical composition is 7-8;

[0184] 9) is the pharmaceutical composition of 1)-8), wherein the sucrose or sorbitol is replaced by mannitol.

[0185] 10) is the pharmaceutical composition according to 1) to 8), wherein the sucrose or sorbitol is replaced by trehalose.

[0186] 11) is the pharmaceutical composition according to 1)-4), wherein the buffer is histidine.

[0187] 12) The pharmaceutical composition according to 1) to 11), further comprising water for injection.

[0188] In some embodiments, the present disclosure provides a pharmaceutical composition comprising any of the aforementioned human insulin analog fusion proteins (e.g., as shown in the amino acid sequence of SEQ ID NO: 19), which comprises or is any one of the following groups 1) to 37):

[0189] 1) 1-200 mg / mL fusion protein;

[0190] about 80 mM or about 40 mM histidine;

[0191] 0.1mg / mL-1mg / mL polysorbate 80 or poloxamer 188;

[0192] about 70 mg / mL sucrose, about 60 mg / mL or about 45 mg / mL sorbitol;

[0193] and the pH of the pharmaceutical composition is 7-8 or 6.5-7.5;

[0194] Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0195] 2) 1-200 mg / mL fusion protein;

[0196] About 80 mM histidine;

[0197] 0.1mg / mL-1mg / mL polysorbate 80;

[0198] about 88 mg / mL trehalose or about 50 mg / mL mannitol;

[0199] and the pH of the pharmaceutical composition is 7-8 or 6.5-7.5;

[0200] Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises or is selected from one or more of benzyl alcohol, phenol and m-cresol;

[0201] 3) 1-200 mg / mL fusion protein, such as about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL;

[0202] About 80 mM histidine;

[0203] about 0.2 mg / mL or about 0.4 mg / mL polysorbate 80;

[0204] about 70 mg / mL sucrose or about 45 mg / mL sorbitol;

[0205] and the pH of the pharmaceutical composition is about 6.5, about 7.0, or about 7.5;

[0206] Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising or selected from about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol;

[0207] 4) 1-200 mg / mL fusion protein, such as about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL;

[0208] About 80 mM histidine;

[0209] about 0.2 mg / mL or about 0.4 mg / mL polysorbate 80;

[0210] about 88 mg / mL trehalose or about 50 mg / mL mannitol;

[0211] and the pH of the pharmaceutical composition is about 6.5, about 7.0, or about 7.5;

[0212] Optionally, the pharmaceutical composition further comprises a bacteriostatic agent selected from about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol;

[0213] 5) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0214] About 80 mM histidine;

[0215] About 0.2 mg / mL polysorbate 80;

[0216] approximately 70 mg / mL sucrose;

[0217] and the pH of the pharmaceutical composition is about 7.0;

[0218] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0219] 6) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0220] About 80 mM histidine;

[0221] About 0.4 mg / mL polysorbate 80;

[0222] approximately 70 mg / mL sucrose;

[0223] and the pH of the pharmaceutical composition is about 7.0;

[0224] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0225] 7) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0226] About 80 mM histidine;

[0227] About 0.2 mg / mL polysorbate 80;

[0228] approximately 70 mg / mL sucrose;

[0229] and the pH of the pharmaceutical composition is about 7.5;

[0230] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0231] 8) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0232] About 80 mM histidine;

[0233] About 0.4 mg / mL polysorbate 80;

[0234] approximately 70 mg / mL sucrose;

[0235] and the pH of the pharmaceutical composition is about 7.5;

[0236] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0237] 9) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0238] About 80 mM histidine;

[0239] About 0.2 mg / mL polysorbate 80;

[0240] approximately 70 mg / mL sucrose;

[0241] and the pH of the pharmaceutical composition is about 7.0;

[0242] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0243] 10) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0244] About 80 mM histidine;

[0245] About 0.4 mg / mL polysorbate 80;

[0246] approximately 70 mg / mL sucrose;

[0247] and the pH of the pharmaceutical composition is about 7.0;

[0248] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0249] 11) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0250] About 80 mM histidine;

[0251] About 0.2 mg / mL polysorbate 80;

[0252] approximately 70 mg / mL sucrose;

[0253] and the pH of the pharmaceutical composition is about 7.5;

[0254] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0255] 12) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0256] About 80 mM histidine;

[0257] About 0.4 mg / mL polysorbate 80;

[0258] approximately 70 mg / mL sucrose;

[0259] and the pH of the pharmaceutical composition is about 7.5;

[0260] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0261] 13) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0262] About 80 mM histidine;

[0263] About 0.2 mg / mL polysorbate 80;

[0264] approximately 70 mg / mL sucrose;

[0265] and the pH of the pharmaceutical composition is about 7.0;

[0266] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0267] 14) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0268] About 80 mM histidine;

[0269] About 0.4 mg / mL polysorbate 80;

[0270] approximately 70 mg / mL sucrose;

[0271] and the pH of the pharmaceutical composition is about 7.0;

[0272] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0273] 15) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0274] About 80 mM histidine;

[0275] About 0.2 mg / mL polysorbate 80;

[0276] approximately 70 mg / mL sucrose;

[0277] and the pH of the pharmaceutical composition is about 7.5;

[0278] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0279] 16) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0280] About 80 mM histidine;

[0281] About 0.4 mg / mL polysorbate 80;

[0282] approximately 70 mg / mL sucrose;

[0283] and the pH of the pharmaceutical composition is about 7.5;

[0284] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0285] 17) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0286] About 80 mM histidine;

[0287] About 0.2 mg / mL polysorbate 80;

[0288] approximately 45 mg / mL sorbitol;

[0289] and the pH of the pharmaceutical composition is about 7.0;

[0290] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0291] 18) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0292] About 80 mM histidine;

[0293] About 0.4 mg / mL polysorbate 80;

[0294] approximately 45 mg / mL sorbitol;

[0295] and the pH of the pharmaceutical composition is about 7.0;

[0296] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0297] 19) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0298] About 80 mM histidine;

[0299] About 0.2 mg / mL polysorbate 80;

[0300] approximately 45 mg / mL sorbitol;

[0301] and the pH of the pharmaceutical composition is about 7.5;

[0302] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0303] 20) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0304] About 80 mM histidine;

[0305] About 0.4 mg / mL polysorbate 80;

[0306] approximately 45 mg / mL sorbitol;

[0307] and the pH of the pharmaceutical composition is about 7.5;

[0308] Optionally, the pharmaceutical composition further comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol;

[0309] 21) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, for example, about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0310] About 80 mM histidine;

[0311] About 0.2 mg / mL polysorbate 80;

[0312] approximately 45 mg / mL sorbitol;

[0313] and the pH of the pharmaceutical composition is about 7.0;

[0314] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0315] 22) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0316] About 80 mM histidine;

[0317] About 0.4 mg / mL polysorbate 80;

[0318] approximately 45 mg / mL sorbitol;

[0319] and the pH of the pharmaceutical composition is about 7.0;

[0320] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0321] 23) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0322] About 80 mM histidine;

[0323] About 0.2 mg / mL polysorbate 80;

[0324] approximately 45 mg / mL sorbitol;

[0325] and the pH of the pharmaceutical composition is about 7.5;

[0326] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0327] 24) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0328] About 80 mM histidine;

[0329] About 0.4 mg / mL polysorbate 80;

[0330] approximately 45 mg / mL sorbitol;

[0331] and the pH of the pharmaceutical composition is about 7.5;

[0332] Optionally, the pharmaceutical composition further comprises about 2.5 mg / mL or about 5 mg / mL phenol;

[0333] 25) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0334] About 80 mM histidine;

[0335] About 0.2 mg / mL polysorbate 80;

[0336] approximately 45 mg / mL sorbitol;

[0337] and the pH of the pharmaceutical composition is about 7.0;

[0338] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0339] 26) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0340] About 80 mM histidine;

[0341] About 0.4 mg / mL polysorbate 80;

[0342] approximately 45 mg / mL sorbitol;

[0343] and the pH of the pharmaceutical composition is about 7.0;

[0344] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0345] 27) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0346] About 80 mM histidine;

[0347] About 0.2 mg / mL polysorbate 80;

[0348] approximately 45 mg / mL sorbitol;

[0349] and the pH of the pharmaceutical composition is about 7.5;

[0350] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0351] 28) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0352] About 80 mM histidine;

[0353] About 0.4 mg / mL polysorbate 80;

[0354] approximately 45 mg / mL sorbitol;

[0355] and the pH of the pharmaceutical composition is about 7.5;

[0356] Optionally, the pharmaceutical composition further comprises about 1 mg / mL or about 3 mg / mL m-cresol;

[0357] 29) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0358] approximately 40 mM histidine;

[0359] approximately 0.4 mg / mL poloxamer 188;

[0360] approximately 60 mg / mL sucrose;

[0361] and the pH of the pharmaceutical composition is about 6.0, about 7.0, or about 7.5;

[0362] Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol, and m-cresol, for example, about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol;

[0363] 30) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0364] approximately 40 mM histidine;

[0365] approximately 0.4 mg / mL poloxamer 188;

[0366] approximately 60 mg / mL sucrose;

[0367] and the pH of the pharmaceutical composition is about 7.0;

[0368] Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol, and m-cresol, for example, about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol;

[0369] 31) about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL, or 150 mg / mL fusion protein, such as about 40 mg / mL, about 60 mg / mL, or about 100 mg / mL;

[0370] approximately 40 mM histidine;

[0371] approximately 0.4 mg / mL poloxamer 188;

[0372] approximately 60 mg / mL sucrose;

[0373] and the pH of the pharmaceutical composition is about 7.5;

[0374] Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol, and m-cresol, for example, about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol;

[0375] 32) is any one of the pharmaceutical compositions of 3-16), wherein polysorbate 80 is replaced by poloxamer 188;

[0376] 33) is any one of the pharmaceutical compositions of 5-16), wherein sucrose is replaced with 80 mg / mL sucrose or 60 mg / mL;

[0377] 34) is any one of the pharmaceutical compositions of 1) to 16), wherein sucrose is replaced with 88 mg / mL trehalose;

[0378] 35) is any one of the pharmaceutical compositions of 1) to 16), wherein sucrose is replaced with 50 mg / mL mannitol;

[0379] 36) is any one of the pharmaceutical compositions of 1) to 35), wherein the pH of the pharmaceutical composition may be about 6.5;

[0380] 37) is any one of the pharmaceutical compositions of 1) to 36), wherein the final volume is 1 mL; when the volume needs to be fixed, the volume is fixed to 1 mL with water for injection.

[0381] The pharmaceutical composition disclosed herein has sufficient drug stability and can be stored stably for a long time.

[0382] In some embodiments, the pharmaceutical composition is stable at 2-8° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or at least 36 months. In some embodiments, the pharmaceutical composition is stable at 25° C. for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the pharmaceutical composition is stable at 40° C. for at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

[0383] The present disclosure provides a method for preparing the aforementioned pharmaceutical composition, comprising the step of dissolving the aforementioned fusion protein.

[0384] To facilitate drug transportation, the pharmaceutical composition of the present disclosure can be further prepared into a lyophilized preparation.

[0385] The present disclosure also provides a lyophilized preparation, characterized in that the lyophilized preparation can form any of the above pharmaceutical compositions after reconstitution.

[0386] The present disclosure also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition as described above.

[0387] The present disclosure provides a reconstitution solution, wherein the reconstitution solution is prepared by reconstitution of the aforementioned lyophilized preparation. In certain embodiments, the reconstitution solution is selected from but not limited to water for injection, physiological saline or glucose solution.

[0388] The present disclosure also provides a product, comprising a container containing the aforementioned pharmaceutical composition, the aforementioned lyophilized formulation, or the aforementioned reconstituted solution. In certain embodiments, the container is a neutral borosilicate glass injection vial. In certain embodiments, the product includes a package insert.

[0389] The present disclosure also provides a pharmaceutical composition or a lyophilized formulation or a reconstituted solution of a lyophilized formulation, which is used as a medicine for treating or alleviating a disease or condition.

[0390] Methods for treating diseases and pharmaceutical uses

[0391] The present disclosure provides pharmaceutical uses of the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation for preparing a drug for preventing, treating or alleviating the aforementioned diseases or symptoms.

[0392] The present disclosure provides uses and methods of the aforementioned pharmaceutical composition, lyophilized preparation, or reconstituted solution of the lyophilized preparation in preventing, treating, or alleviating diseases or symptoms.

[0393] The present disclosure provides a method for preventing, treating, or alleviating a disease or symptom, comprising administering to a patient or subject a preventively and / or therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized preparation or a reconstituted solution of the lyophilized preparation.

[0394] In some embodiments, the method comprises administering an effective amount of a fusion protein or insulin analog of the present disclosure to a subject in need thereof for preventing, treating, or ameliorating a disease or symptom.

[0395] In some embodiments, the disease or symptom is selected from non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus or other metabolic diseases. The subject in need thereof has not been treated or is treated with oral medications (such as sulfonylureas, metformin, thiazolidinediones such as pioglitazone, α-glucosidase inhibitors such as acarbose) and / or non-insulin injectables (including incretin-based therapies such as DPP-4 inhibitors and GLP-1R agonists).

[0396] In some embodiments, the disease or condition is selected from diabetes, obesity, dyslipidemia, and / or metabolic syndrome.

[0397] In some embodiments, the disease or condition is a diabetic complication, or diabetes-related heart disease, stroke, nephropathy, retinopathy, neuropathy, or renal disease.

[0398] In some embodiments, the disease or condition is selected from hyperglycemia, type II diabetes, impaired glucose tolerance, type I diabetes, obesity, syndrome X, or dyslipidemia.

[0399] In some embodiments, methods are provided for administering an effective amount of a fusion protein or insulin analog of the present disclosure to a subject in need thereof for lowering blood glucose.

[0400] The pharmaceutical composition of the present disclosure can be used to treat patients in need of such treatment by parenteral administration. The parenteral administration route can be selected from subcutaneous injection, intramuscular injection or intravenous injection.

[0401] the term

[0402] To facilitate understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure pertains. The three-letter and one-letter amino acid codes used herein are as described in J. Biol. Chem, 243, p3558 (1968).

[0403] "Insulin" includes naturally occurring insulin (e.g., human insulin), which is composed of two polypeptide chains, referred to as A and B chains, comprising 21 and 30 amino acid residues, respectively, connected by two cysteine ​​disulfide bonds. The A chain is exemplified by SEQ ID NO: 29 of the present disclosure, and the B chain is exemplified by SEQ ID NO: 30 of the present disclosure.

[0404] "Insulin analogs" include polypeptides that have a molecular structure that can be formally derived from naturally occurring insulin (e.g., human insulin structure) by removing and / or replacing (displacing) one or more amino acid residues present in natural insulin and / or by adding one or more amino acid residues. The added and / or substituted amino acid residues can be codable amino acid residues or other naturally occurring amino acid residues or purely synthetic amino acid residues.

[0405] An "insulin A chain analogue" refers to an insulin A chain analogue that has one or more amino acid substitutions, deletions and / or extensions (additions) relative to the A chain of human insulin.

[0406] An "insulin B chain analogue" refers to an insulin B chain analogue that has one or more amino acid substitutions, deletions and / or extensions (additions) of the B chain relative to the B chain of human insulin.

[0407] "Glucose-responsive insulin" (GRI) can regulate the release and action of insulin according to blood sugar levels, thereby reducing the incidence of hypoglycemia and making blood sugar control more stable.

[0408] The term "insulin receptor agonist" refers to a protein that binds to and activates the insulin receptor, resulting in a decrease in blood glucose levels and / or inhibition of hepatic glucose excretion, and is characterized in that it can be tested and measured using known techniques (such as those shown in the studies described in the Examples of this disclosure). In some embodiments, the insulin receptor agonist is formed by directly or indirectly linking an insulin A chain analog to an insulin B chain analog (e.g., via a linker).

[0409] "Sequence" should generally be understood to include both the relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the sequence, unless the present disclosure requires further limited explanation.

[0410] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0411] "About" and "approximately" refer to values ​​within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" or "substantially comprising" can mean a range of up to 20%, such as between 1% and 15%, between 1% and 10%, between 1% and 5%, between 0.5% and 5%, between 0.5% and 1%, and in this disclosure, each instance of a number or numerical range preceded by the term "about" also includes embodiments of the given number. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for that specific value.

[0412] "Buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control pH in an appropriate range include acetate, succinate, histidine, phosphate, citrate, and other organic acid buffers.

[0413] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. Preferred are histidine hydrochloride buffers or histidine acetate buffers. Histidine acetate buffers are prepared from histidine and acetic acid, and histidine hydrochloride buffers are prepared from histidine and histidine hydrochloride, or histidine and hydrochloric acid.

[0414] "Phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. Preferably, the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0415] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient to exert its biological activity.

[0416] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0417] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0418] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0419] The pharmaceutical compositions disclosed herein can achieve a stable effect in which the fusion protein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. For example, the pharmaceutical composition substantially retains its physical and chemical stability as well as its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Various analytical techniques are currently available for measuring protein stability after storage at a selected temperature for a selected period of time.

[0420] Stable pharmaceutical fusion protein formulations are formulations in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, at least 2 years, and up to 3 years. In addition, stable liquid formulations include those that exhibit desired characteristics after being stored at 25°C for 1 month, 3 months, 6 months, or at 40°C for 1 month. Typical acceptable standards for stability are as follows: as measured by SEC-HPLC, typically no more than about 10%, preferably no more than about 5%, of the fusion protein degrades. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed, and typically no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0421] A fusion protein "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0422] If the fusion protein does not show significant chemical changes, then the fusion protein "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0423] An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the fusion protein at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0424] "Administer," "give," and "treat," as they apply to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0425] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein or pharmaceutical compositions thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the treated subject or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other health care professionals to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating the symptoms of the target disease in a certain subject, it should alleviate the symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0426] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0427] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0428] "Subject" or "patient" refers to a mammal, particularly a primate, and especially a human.

[0429] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be construed to have an inclusive sense rather than an exclusive or exhaustive sense; that is, in the sense of "including but not limited to."

[0430] The equipment and methods used in the testing process are as follows:

[0431] Appearance:

[0432] Using the visual method, wipe the sample bottle clean and observe the sample color, clarity and visible foreign matter on a clarity tester with a white background and a black background under a light intensity of 1000-1500lx.

[0433] Appearance inspection instrument: Jingtuo Instrument YB-2A clarity tester.

[0434] SEC size exclusion chromatography:

[0435] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0436] SEC monomer content percentage = A monomer / A total * 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas.)

[0437] SEC instrument: Agilent 1260 or Waters ACQUITY ARC; Column: Waters, XBrige Protein BEH SEC SEC (300×7.8mm 3.5μm)

[0438] NR-CE capillary gel electrophoresis:

[0439] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0440] Non-reduced CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the main peak in the sample, and A total is the sum of all peak areas.

[0441] CE test instrument: Sciex model PA800 plus

[0442] icIEF imaging capillary isoelectric focusing electrophoresis:

[0443] A technique for separating proteins based on their isoelectric points (pI).

[0444] icIEF main peak content percentage = main peak area / total area*100% (total area is the sum of the areas of acidic peak, main peak and basic peak).

[0445] The instrument used for icIEF determination was manufactured by Protein Simple, model number: Muarice.

[0446] Protein concentration determination:

[0447] Protein concentration was determined using a UV-visible spectrophotometer (Nano Drop 2000) with a pathlength of 1 mm.

[0448] Exemplary fusion protein pharmaceutical composition (preparation) preparation process

[0449] Step 1: A human insulin analog fusion protein (e.g., the amino acid sequence of SEQ ID NO: 19) is mixed with the following excipients in the prescribed amounts to prepare a stock solution containing the fusion protein. After filtration, a sterility control sample is taken. The stock solution is filtered through a 0.22 μm filter cartridge, and the filtrate is collected.

[0450] Step 2: Adjust the filling volume to 1.15 mL, fill the filtrate into a 2 mL vial, add a stopper, and use online weighing during the filling process to control the filling volume difference within the qualified range.

[0451] Step 3: Turn on the capping machine, add aluminum caps, and start capping.

[0452] Step 4: Visual inspection to confirm that the product has no defects such as inaccurate filling quantity. Print and apply vial labels; print carton labels, folding cartons, packing, and affixing box labels. BRIEF DESCRIPTION OF THE DRAWINGS

[0453] FIG1 is a graph showing the hypoglycemic effect of a single subcutaneous injection of fusion protein 1 in a type I diabetic rat model induced by STZ.

[0454] Figure 2 shows the hypoglycemic effect of a single subcutaneous injection of LY3209590, fusion protein 2, and fusion protein 9 in a type I diabetic rat model induced by STZ. DETAILED DESCRIPTION

[0455] The present disclosure is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0456] Experimental methods in the examples disclosed herein, where specific conditions are not specified, generally followed conventional conditions or the conditions recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0457] Example

[0458] The preparation and purification methods of the human insulin analog-Fc fusion protein in this application have been described in the patent document with application number WO2023174370, and the entire contents of the aforementioned application document can be incorporated into the present disclosure.

[0459] Example 1. Design of human insulin analogs and their Fc fusion proteins

[0460] This embodiment provides a human insulin analog, the general formula from N-terminus to C-terminus is: B1-L1-A1.

[0461] Among them, B1 is a human insulin B chain analogue with the sequence:

[0462] FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1), wherein X1 is an amino acid residue selected from Asn, Gly or Lys, X2 is an amino acid residue selected from Ser or Glu, X3 is an amino acid residue selected from Tyr, His or Glu, X4 is an amino acid residue selected from His, Arg, Leu or Glu, and X5 is an amino acid residue selected from Phe or His;

[0463] A1 is an insulin A chain analog, having the sequence: GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO: 2), wherein Z1 is selected from an amino acid residue of Thr, His or Glu, Z2 is selected from an amino acid residue of Tyr, Asp, Ser or Glu, and Z3 is selected from an amino acid residue of Gly or Asn;

[0464] L1 is GGGGGGSGGGG (SEQ ID NO:3) or GGGGGGSGGGG (SEQ ID NO:52).

[0465] The sequence of an exemplary human insulin analog is as follows:

[0466] >Human insulin analog 1

[0467] >Human insulin analogue 2

[0468] >Human insulin analogue 3

[0469] >Human insulin analogue 4

[0470] >Human insulin analogue 5

[0471] >Human insulin analog 6

[0472] Furthermore, a fusion protein of the above human insulin analog and Fc is provided, and the fusion of the two is achieved through a linker as shown in Table 1.

[0473] Table 1. Linker sequences of human insulin analogs fused to Fc

[0474] The Fc is human IgG2 Fc, and the amino acid sequence is as follows:

[0475] >IgG2 Fc1

[0476] >IgG2 Fc2

[0477] The amino acid sequence of an exemplary fusion protein is as follows:

[0478] >Fusion protein 1 (insulin analog 1+L2+IgG2 Fc1)

[0479] >Fusion protein 2 (insulin analog 2+L2+IgG2 Fc1)

[0480] >Fusion protein 3 (insulin analog 3+L2+IgG2 Fc1)

[0481] >Fusion protein 4 (insulin analog 4+L2+IgG2 Fc1)

[0482] >Fusion protein 5 (insulin analog 1+L2+IgG2 Fc1)

[0483] >Fusion protein 6 (insulin analog 5+L2+IgG2 Fc1)

[0484] >Fusion protein 7 (insulin analog 1+L2+IgG2 Fc1)

[0485] >Fusion protein 8 (insulin analog 1+L2+IgG2 Fc1)

[0486] >Fusion protein 9 (insulin analog 6+L2+IgG2 Fc1)

[0487] In addition, LY3209590 is an ultra-long-acting insulin analog developed by Eli Lilly and Company in the United States for once-weekly administration. It is currently in Phase III clinical trials. Its amino acid sequence is:

[0488] The underlined sequences are linker sequences.

[0489] The wild-type human insulin sequence is as follows:

[0490] >A Chain

[0491] >B Chain

[0492] Example 2. Preparation, characterization and identification of human insulin analog-Fc fusion protein

[0493] 1. Preparation

[0494] 1) HEK-293 cell expression

[0495] The correct plasmid was extracted using an endotoxin-free midi kit (Promega, A2495). After sequencing, cells were prepared for transfection and protein production. HEK293 suspension cells were passaged regularly using Freestyle 293 medium (Invitrogen, cat No. 12338026) and passaged to a density of 6 × 10 cells per day before transfection. 5 cells / mL. On the day of transfection, the cell viability and density were measured to ensure that the cell viability was >90%, and the cell density was adjusted to 1.1×10 6Prepare plasmid DNA at 1 μg DNA / mL per cell. Dilute the DNA and corresponding 293-fectin (Invitrogen, cat. No. 12347019) in Opti-MEM (Invitrogen, cat. No. 51985-034), gently shake to mix, and let it stand at room temperature for 5 minutes. Gently shake the diluted DNA and liposomes to mix. After standing at room temperature for 20 minutes, slowly add them to the HEK293 cells to be transfected, gently swirling the cell culture flask. The cells are then incubated in a 37°C shaker (5% CO2, 135 rpm, 75% humidity) in suspension. Five days after transfection, collect the cell culture supernatant, centrifuge at 4000 rpm for 20 minutes, and filter the supernatant through a 0.45 μm filter.

[0496] 2) ExpiCHO cell expression

[0497] ExpiCHO suspension cells were passaged regularly using ExpiCHO Expression Medium (Invitrogen, Cat. No. A2910002) and transfected using the ExpiCHO Expression System (Cat. No. A29133) at a transfection ratio of 1 μg DNA / mL. Following transfection, cells were cultured in a 32°C shaker (5% CO2, 110 rpm, 75% humidity) using the "High protocol" specified in the instructions. Cell culture supernatants were harvested 10-12 days after transfection, centrifuged at 4000 rpm for 20 minutes, and filtered through a 0.45 μm filter.

[0498] 3) Expression level detection

[0499] The OD280 of purified human insulin analog-Fc fusion proteins was measured by UV absorption, and the expression levels of each fusion protein in different expression systems were estimated based on this. The results showed that fusion proteins 1 to 9 were all expressed in the HEK293 expression system, with some fusion proteins showing excellent yields. For example, fusion protein 1 expressed 119 mg / L, fusion protein 4 expressed 116 mg / L, fusion protein 6 expressed 132 mg / L, fusion protein 7 expressed 110 mg / L, and fusion protein 8 expressed 119 mg / L. In the ExpiCHO transient expression system, the expression level of fusion protein 1 exceeded 400 mg / L.

[0500] 4) Purification

[0501] Protein-A affinity chromatography:

[0502] The supernatant from cell cultures expressing the Fc fusion protein was collected by high-speed centrifugation and filtered through a 0.22 μm filter. A Protein-A (GE, Cat: 17-5474-99) affinity column was regenerated with 5 column volumes of 0.1 M NaOH (Sigma, Cat: 71687-500g), followed by washing and equilibration with 5 column volumes of 1× PBS buffer (pH 7.4) (Sangon Biotech (Shanghai) Co., Ltd., Cat. E607016-0500). The supernatant was loaded at a low flow rate to facilitate binding, controlling the flow rate to maintain a retention time of at least 1 minute. After binding was complete, the column was rinsed with 5-10 column volumes of 1× PBS buffer (pH 7.4) until the UV absorbance returned to baseline. Afterwards, the sample was eluted using 0.1M glycine (pH 3.0) (Sigma: Cat.410225-250G) buffer, and the elution peak was collected according to UV detection. The eluted product was quickly adjusted to pH 7-8 using 1M Tris-HCl (pH 7.5) (Vetec, Cat. V900483) and temporarily stored. For the eluted product, solution replacement can be performed using methods well known to those skilled in the art, such as ultrafiltration and concentration using an ultrafiltration tube, and solution replacement to the desired buffer system, or replacement with the desired buffer system using molecular exclusion (such as G-25 desalting). The final storage system for the human insulin analog-Fc fusion protein is 1× PBS buffer, pH 7.4.

[0503] Anion chromatography:

[0504] In the first step, the sample was diluted with equilibration buffer (20 mM Tris pH 8.0, Vetec, Cat. V900483) to a conductivity below 3 mS / cm. A Q HP (GE, Cat. 17-1014-01) anion chromatography column was regenerated with 5 column volumes of 0.5 M NaOH (Sigma, Cat. 71687-500g) and then washed and equilibrated with 15 column volumes of 20 mM Tris buffer, pH 8.0 (Vetec, Cat. V900483). The supernatant was loaded at a low flow rate to facilitate binding, controlling the flow rate to maintain a retention time of at least 2 minutes. After binding was complete, the column was flushed with 10 column volumes of 20 mM Tris buffer, pH 8.0 (Vetec, Cat. V900483) until the UV absorbance returned to baseline. Elution was performed with a gradient of 0-100% using 20 mM Tris pH 8.0 (Vetec, Cat. V900483) 0.5 M NaCl (Vetec, Cat. V900058); HPLC was performed according to SEC-HPLC (Column: TSKgel G3000SW XL, 5 μm, Cat. 008541) for purity, and the eluted peak was collected and tested. The eluted product can be subjected to solution exchange using methods familiar to those skilled in the art, such as ultrafiltration and concentration using an ultrafiltration tube and solution exchange into the desired buffer system, or molecular exclusion (such as G-25 desalting) to replace the desired buffer system. The final storage buffer system for the human insulin analog-Fc fusion protein is 1× PBS buffer, pH 7.4.

[0505] 2. Characterization and Identification

[0506] Size exclusion chromatography (SEC-HPLC): An Agilent 1260 Infinity HPLC system was used, with a Tosoh column (TSGel-G3000SWXL, 7.8 mm × 300 mm). Prior to injection, the column and system were equilibrated with 2× PBS buffer containing 5% isopropanol. Then, 15 μg of the sample was injected and eluted with the same buffer. The method duration was 20 min, and the method flow rate was 1 mL / min.

[0507] Ultra-High-Performance Liquid Chromatography-Mass Spectrometry: Characterization was performed using an Agilent 1290-6530 ultra-high-performance liquid chromatography-mass spectrometry system. A Separator-C4 column (particle size 3 μm, column dimensions: 2.1 mm × 50 mm) was used. Mobile phase A consisted of an aqueous solution containing 0.1% formic acid, and mobile phase B consisted of acetonitrile containing 0.1% formic acid. The flow rate was 1 mL / min, and the column temperature was maintained at 60°C. The chromatographic gradient used is shown in Table 2. Mass spectrometer parameters were set to: 1 GHz mode, capillary voltage: 5000 V, desolvation gas temperature: 350°C, gas flow rate: 12 L / min, fragmentor voltage: 380 V, and MS acquisition range: 100 m / z–4000 m / z.

[0508] Table 2. Chromatographic gradient

[0509] The data in Table 3 show that the fusion proteins 1 to 9 all have good purity and are in line with the expected molecular weight after purification.

[0510] Table 3. SEC-HPLC purity and molecular weight

[0511] The following examples test the biological activities of the fusion proteins disclosed herein.

[0512] Example 3. Human insulin receptor type B (hIR-B) autophosphorylation detection experiment

[0513] The activity of different human insulin analog-Fc fusion proteins in promoting the autophosphorylation of human insulin receptor B subtype was investigated and compared in CHO-K1 cells overexpressing human insulin receptor B subtype. The EC obtained in this test system was calculated. 50 The activity of each fusion protein in promoting the autophosphorylation of human insulin receptor B subtype was evaluated. The lower EC 50 The higher the value, the higher the in vitro activity.

[0514] Experimental method: 18 hours before the start of the experiment, CHO-K1-hIRB cells (Shanghai Huiyuan, CHO-K1-hIRB-M-20170706) were used to plate 96-well plates. 4 100 μL growth medium (F-12K medium, 1× penicillin-streptomycin containing 10% FBS and 4 μg / mL blasticidin) was added to each well and cultured overnight at 37°C in 5% CO2. On the morning of the experiment, human insulin analog-Fc fusion protein was diluted as required in 50 μL starvation medium (F-12K medium, 1× penicillin-streptomycin containing 0.1% BSA and 4 μg / mL blasticidin). After discarding the old medium, the cells were incubated with starvation medium containing human insulin analog-Fc fusion protein at 37°C in 5% CO2 for 20 minutes. The medium was then discarded and the cells were lysed with the cell lysis buffer in the kit (IR-beta phospho-Y1150 / 1151 kit, Cisbio, batch number: 63ADK016PEG) and incubated at 37°C for 30 minutes. 16 μL of lysate was then transferred to a 96-well plate and 4 μL of the prepared antibody mixture (containing Phospho-Y1150 / 1151) was added. InsuLin Receptor beta Cryptate antibody and Phospho InsuLin Receptor beta d2 antibody) and incubate at room temperature for 4 hours.

[0515] Data processing: Signals were read using a Tecan microplate reader with an excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The signal ratio (665 nm / 620 nm × 10,000) was calculated and the EC was obtained by nonlinearly fitting the signal ratio to the sample concentration using a four-parameter equation in GraphPad Prism 6. 50 The values ​​are shown in Table 4. Human insulin used as a control in this example and in the following examples was purchased from Sigma-Aldrich.

[0516] Table 4. Type B insulin receptor autophosphorylation activity of human insulin analog-Fc fusion proteins

[0517] The results in Table 4 show that the fusion proteins disclosed herein all have certain activity in activating autophosphorylation of insulin receptor B subtype, EC 50 The values ​​show that the activity of all fusion proteins is significantly weaker than that of native human insulin, and some fusion proteins have activity comparable to or slightly stronger than LY3209590 in activating insulin receptor B subtype autophosphorylation. This indicates that the disclosed fusion proteins are all human insulin receptor agonists, but their activity is weakened compared to native human insulin, which allows for a safer dosing window in clinical practice and can reduce side effects such as hypoglycemia in patients.

[0518] Example 4. Detection of AKT phosphorylation activity in C2C12 mouse myoblasts

[0519] After insulin analogs bind to the insulin receptor, they activate a series of downstream signaling pathways. Among them, stimulation of AKT phosphorylation is a major signaling pathway for insulin and its analogs to produce hypoglycemic effects. Therefore, the activity of different human insulin analog-Fc fusion proteins in stimulating AKT phosphorylation was investigated in C2C12 mouse myoblasts and compared. The EC values ​​of each fusion protein in this test system were calculated. 50 The phosphorylation activity of AKT in mouse myoblasts stimulated by each fusion protein was evaluated. The lower EC 50 A higher value means higher activity.

[0520] Experimental method: 18 hours before the start of the experiment, C2C12 cells (Procell, CL-0044) were plated in 96-well plates. 4 100 μL of growth medium (DMEM medium, 1× penicillin-streptomycin with 10% FBS) was added to each well and incubated at 37°C in 5% CO2 overnight. On the morning of the experiment, human insulin analog-Fc fusion protein was diluted as required in 50 μL of starvation medium (DMEM medium, 1× penicillin-streptomycin without FBS). After discarding the old medium, the cells were incubated with starvation medium containing human insulin analog-Fc fusion protein in a 37°C incubator with 5% CO2 for 20 minutes. The medium was then discarded and the cells were lysed with the cell lysis buffer in the kit (AKT phospho-S473 kit, Cisbio, 64AKSPEG) and incubated at 37°C for 30 minutes. 16 μL of lysate was then transferred to a 96-well plate and 4 μL of the prepared antibody mixture (containing Phospho-AKT Eu Cryptate antibody and Phospho-AKT d2 antibody) was added and incubated at room temperature for 4 hours. Using the data processing method in Example 3, we can get EC 50 Values ​​are shown in Table 5.

[0521] Table 5. Human insulin analog-Fc fusion protein stimulates AKT phosphorylation activity in C2C12 mouse myoblasts

[0522] The results showed that the fusion proteins disclosed in the present invention have certain activity in activating the phosphorylation of insulin receptor AKT. 50 The values ​​showed that the activity of all fusion proteins was significantly weaker than that of native human insulin, and the activity of some fusion proteins was comparable to that of LY3209590. This indicates that all fusion proteins have the activity of significantly stimulating AKT phosphorylation in mouse myoblasts, but their activity is weaker than that of native human insulin.

[0523] Example 5. Experiment on promoting proliferation of human MCF-7 (human breast cancer cells)

[0524] The ability of some human insulin analog-Fc fusion proteins disclosed herein to activate downstream signaling pathways and promote cell proliferation was investigated in human MCF-7 breast cancer cells. Numerous studies have shown that, in addition to their metabolic activities, such as inducing blood sugar reduction, insulin and insulin analogs also activate other downstream signaling pathways, thereby promoting cell proliferation. This cell proliferation-promoting activity is closely related to the tumorigenesis-inducing effects of insulin and its analogs. In particular, the late-stage development of Novo Nordisk's fast-acting insulin analog, B10 Asp insulin, was halted after significant tumorigenesis was observed in a rat model. Therefore, the purpose of this study was to evaluate the cell proliferation-inducing ability of the fusion proteins disclosed herein using MCF-7 human breast cancer cells. Significantly reduced cell proliferation-promoting ability suggests a lower risk of tumorigenesis later in life. Studies have shown that this cell proliferation-promoting ability of insulin or its analogs is associated with the expression of IGF-1R on the surface of some cells. Insulin or its analogs react with IGF-1R, promoting cell proliferation and potentially inducing tumor development.

[0525] Experimental method: Before the experiment, MCF-7 cells (Procell, CL-147) were resuspended in DMEM medium containing 0.5% FBS and counted. 100 μL of cell suspension was added to each well of a 96-well black cell culture plate with a clear bottom. The density was 5×10 3Cells / well were cultured overnight at 37°C and 5% CO2. On the morning of the experiment, human insulin analog-Fc fusion protein was diluted as required in 50 μL starvation medium (DMEM medium containing 0.5% FBS). After discarding the old medium, the cells were incubated with starvation medium containing human insulin analog-Fc fusion protein at 37°C and 5% CO2 for 3 days. After the incubation, CellTiter-Glo detection solution (50 μL) was added to the 96-well plate and shaken at room temperature for 2 minutes. After being placed in the dark for 10 minutes, the fluorescence value was read using a Tecan microplate reader and the EC was calculated. 50 Values ​​are shown in Table 6.

[0526] Table 6. Human insulin analog-Fc fusion protein promoting MCF-7 proliferation activity

[0527] The results showed that in multiple parallel experiments (Table 6 shows one of them), fusion protein 1 had a weaker activity in stimulating MCF-7 cell proliferation than natural human insulin. Surprisingly, fusion protein 1 had a significantly lower activity in stimulating MCF-7 human breast cancer cell proliferation than LY3209590, suggesting that the fusion protein has a potential lower risk of inducing tumor formation. At the same time, other fusion proteins 2 to 9 disclosed in the present invention also showed lower cell proliferation activity on MCF-7 than human insulin. For example, the EC value of fusion protein 2 in promoting MCF-7 proliferation activity was 50 EC of LY3209590 50 About 2 times the value.

[0528] Example 6. Detection of Pharmacokinetic Properties of Human Insulin Analog-Fc Fusion Protein

[0529] In this example, SD rats were used as an animal model to study the pharmacokinetic properties of fusion protein 1 after a single subcutaneous or intravenous injection in normal male SD rats.

[0530] Experimental Methods: Male SD rats weighing 170-200 g and aged 5-7 weeks (purchased from Vital River) were selected. Fusion protein 1 was prepared in 1× PBS buffer and injected into the rats at a dose of 30 nmol / kg via the tail vein or dorsal subcutaneous injection. After administration, the rats were not fasted. Orbital blood was collected at 15 minutes, 1 hour, 2 hours, 4 hours, and on days 1, 3, 5, 8, 11, 14, 21, and 25 after administration. EDTA anticoagulation and centrifugation were used to obtain plasma for in vivo pharmacokinetic analysis. Pharmacokinetic analysis was performed using an enzyme-linked immunosorbent assay (ELISA). A multiwell plate was coated with a capture antibody, anti-human IgG-Fc (I2136, Sigma). A polyclonal insulin antibody (337E2A20, Invitrogen) was added, which specifically bound to the analyte. The concentration of the analyte in plasma was then quantified using a detection antibody, anti-rabbit IgG peroxidase (A0545, Sigma). The standard curve and samples used in the assay were prepared using 1% rat plasma (EDTA). The results are shown in Tables 7 and 8.

[0531] Table 7. Relationship between blood concentration and time of fusion protein 1 in SD rats

[0532] Table 8. Pharmacokinetic properties of fusion protein 1 in SD rats

[0533] The results showed that fusion protein 1 had ultra-long in vivo pharmacokinetic properties after a single intravenous / subcutaneous injection in normal rats, with a half-life of about 4.5 days, which means that the fusion protein may have a lower clinical dosing frequency and a longer dosing interval.

[0534] Example 7. Pharmacodynamic study of human insulin analog-Fc fusion protein in type 1 diabetic rat model

[0535] In this example, streptozotocin (STZ)-induced rats were used as an animal model of type I diabetes to investigate the hypoglycemic effect and duration of some human insulin analog-Fc fusion proteins after a single subcutaneous injection, in order to comprehensively evaluate the hypoglycemic activity and in vivo pharmacokinetic properties.

[0536] Experimental Methods: Eight-week-old male Sprague-Dawley rats (purchased from Weitonglihua) weighing approximately 350 grams were selected and fed for one week. A citric acid / sodium citrate buffer solution with a pH range of 4.2-4.5 was prepared. STZ was dissolved in this buffer to a 1% (w / v) concentration. STZ was then injected intraperitoneally based on the rat's actual fasting body weight at a dose of 60 mg / kg per rat. Fasting blood glucose was measured on the third day after STZ injection. Successful modeling was considered when the measured blood glucose level was above 15 mM.

[0537] Human insulin analog-Fc fusion protein was prepared to the corresponding concentration using 1×PBS buffer, pH 7.4. Before administration, the blood glucose of each type I diabetic rat was tested, and rats with blood glucose values ​​between 20-30 mM were randomly divided into groups to ensure that the mean blood glucose and standard deviation of each group of 3 or 6 rats were equivalent. After grouping, the corresponding human insulin analog-Fc fusion protein was administered subcutaneously to each group of rats based on their body weight. After administration, the rats were allowed to eat and drink normally, and the blood glucose levels of the animals were continuously monitored at 1 h, 2 h, 4 h, 24 h, 48 h after administration until the blood glucose returned to the blood glucose level before administration, and a graph showing the corresponding blood glucose value changes over time was drawn.

[0538] In the STZ-induced type 1 diabetic rat model, the time-dependent changes in blood glucose concentration following a single subcutaneous injection of fusion protein 1 (dose: 90 nmol / kg) are shown in Figure 1; the time-dependent changes in blood glucose concentration following LY3209590, fusion protein 2, and fusion protein 9 (dose: 30 nmol / kg) are shown in Figure 2. The results showed that fusion protein 1, fusion protein 2, and fusion protein 9 all had a prolonged hypoglycemic effect, with the hypoglycemic effect of fusion protein 1 lasting at least 300 hours after a single subcutaneous injection.

[0539] Example 8. Pharmacodynamic Study of Human Insulin Analog-Fc Fusion Protein in the db / db Type 2 Diabetes Mouse Model

[0540] This example uses the db / db type 2 diabetic mouse model to investigate the improvement of glycated hemoglobin after multiple subcutaneous injections of some human insulin analog-Fc fusion proteins, in order to comprehensively evaluate the hypoglycemic activity and in vivo pharmacokinetic properties.

[0541] Experimental method: 50 db / db mice were purchased and adapted to the environment for 5 days. Animals with random blood glucose levels higher than 20mmol / L were selected for further group experiments. The experiment was divided into 4 groups, namely the model control group (solvent control group, ingredients: 0.08M histidine, 70g / L sucrose, 0.4g / L polysorbate 80), fusion protein 1-low dose group, fusion protein 1-medium dose group and fusion protein 1-high dose group, with 10 animals in each group, a total of 40 animals. The drug was administered on the first day after grouping, once a week, for a total of 4 times, and the administration method was subcutaneous injection. At the end of the experiment, the glycated hemoglobin of all mice was tested. The detection method was to take 0.4mL of blood from the animal through the abdominal main vein, inject it into an EDTA anticoagulant tube, and directly detect the glycated hemoglobin on the machine. The results are shown in Table 9.

[0542] Table 9. Animal Glycated Hemoglobin Results (%)

[0543] Note: The results in the table are expressed as mean ± SD; “*” represents p ≤ 0.05 compared with the model control group.

[0544] The results showed that the glycated hemoglobin level in the model control group was 11.27±0.87%, while the glycated hemoglobin levels in the low, medium, and high dose groups of fusion protein 1 were 11.01±0.86%, 10.58±0.44% (p=0.0481), and 10.41±0.45% (p=0.0171), respectively. The medium and high dose groups showed statistically significant differences compared to the model control group, indicating a dose-response relationship among the three dose groups. These results suggest that the medium and high doses of fusion protein 1 significantly reduced glycated hemoglobin levels in db / db mice, while the low dose group had no significant effect, indicating a dose-response relationship among the three dose groups.

[0545] Example 9. Pharmacokinetic properties of human insulin analog-Fc fusion protein in Bama pigs

[0546] In this example, Bama pigs were used as an animal model to study the pharmacokinetic properties of fusion protein 1 and LY3209590 after a single subcutaneous injection in Bama pigs.

[0547] Experimental Methods: Male Bama pigs weighing 13-18 kg and aged 4-6 months were selected (Yibin Hengshu Biotechnology Co., Ltd.). Fusion protein 1 and LY3209590 were prepared in a vehicle (80 mM histidine, 70 g / L sucrose, 0.4 g / L Tween 80) and 1× PBS buffer, respectively. The animals were subcutaneously injected at doses of 5 nmol / kg and 2 nmol / kg, respectively. Approximately 1 mL of venous blood was collected without anticoagulation at 1, 4, 8, 24, 48, 72, 96, 120, 144, and 168 hours after administration. Whole blood samples were stored in an ice box before centrifugation at approximately 1800 × g for 10 minutes at 2 to 8°C. Serum was separated into two tubes (approximately 100 μL in one tube and the remaining serum in the other tube) and stored below -66°C for in vivo pharmacokinetic analysis. The pharmacokinetic characteristics analysis was performed using a method similar to Example 6. The specific pharmacokinetic parameters are shown in Table 10.

[0548] Table 10. Pharmacokinetic properties of fusion protein 1 and LY3209590 in Bama pigs

[0549] The results showed that fusion protein 1 exhibited exceptionally long in vivo pharmacokinetic properties after a single subcutaneous injection in Bama pigs. At the doses listed in Table 10, the drug half-life (t1 / 2) did not significantly change with dose. Compared to LY3209590, fusion protein 1 exhibited a significantly prolonged half-life of approximately 120 hours, suggesting that this fusion protein has a clinically lower dosing frequency and a longer dosing interval.

[0550] The present disclosure provides a novel fusion protein of a human insulin analog and an IgG Fc region, which can be used to treat metabolic diseases such as type I diabetes or type II diabetes. The fusion protein has an extremely long in vivo pharmacokinetic profile, demonstrates a good and sustained hypoglycemic effect in an STZ-induced type I diabetes rat model and a db / db type II diabetes mouse model, has excellent physical and chemical stability and high expression yield, and exhibits significantly lower in vitro cell proliferation activity compared to native human insulin, suggesting a potentially good safety profile and low medication risk, potentially meeting clinical demand for ultra-long-acting insulin products.

[0551] The human insulin analog-Fc fusion protein used in the following examples is the aforementioned fusion protein-1, and its amino acid sequence is shown in SEQ ID NO:19.

[0552] Example 10. Screening of buffer systems and pH values ​​for human insulin analog-Fc fusion protein preparations

[0553] The following buffer was prepared to prepare a fusion protein-1 preparation with a protein concentration of 100 mg / mL. High temperature (40° C.) and repeated freeze-thaw and shaking stability studies were performed. The experimental results are shown in Table 11.

[0554] F1: 30 mM acetate-histidine, pH 5.0

[0555] F2: 30 mM acetate-histidine, pH 5.5

[0556] F3: 30 mM acetate-histidine, pH 6.0

[0557] F4: 30 mM acetate-histidine, pH 6.5

[0558] F5: 80 mM histidine, pH 7.0

[0559] Table 11. Buffer system and pH value screening results

[0560] Experimental results:

[0561] Appearance results showed that after one week at 40°C, formulations F2 (acetate-histidine buffer, pH 5.5), F3 (acetate-histidine buffer, pH 6.0), and F4 (acetate-histidine buffer, pH 6.5) developed numerous visible particles. After four weeks at 40°C, formulation F1 (acetate-histidine buffer, pH 5.0) also developed numerous visible particles, while formulation F5 (80 mM histidine buffer, pH 7.0) showed no visible particles. These results demonstrate that histidine buffer is superior to other buffer systems.

[0562] SEC results showed no significant decrease in monomer purity across all formulations after five freeze-thaw cycles and three days of shaking. However, after storage at 40°C, monomer purity decreased across all formulations. However, after four weeks at 40°C, F5 (histidine buffer, pH 7.0) showed a smaller decrease, approximately 1.8%, compared to F1, demonstrating improved stability.

[0563] NR-CE results showed that after five freeze-thaw cycles and three days of shaking, the purity of the main NR-CE peak for each formulation did not decrease significantly. After storage at 40°C, the purity of the main NR-CE peak for each formulation decreased. After four weeks at 40°C, the purity of F5 (histidine buffer, pH 7.0) decreased less significantly than F1, by approximately 3.9%, indicating better stability.

[0564] The iCIEF results showed that after five freeze-thaw cycles and three days of shaking, the purity of the iCIEF main peak of each prescription did not show a significant decrease.

[0565] In summary, the appearance, SEC and NR-CE results showed that F5 (histidine buffer, pH 7.0) had good stability.

[0566] Example 11. pH and excipient screening in human insulin analog-Fc fusion protein preparations

[0567] The following different formulations were prepared to produce a 100 mg / mL formulation of fusion protein-1 and to investigate its stability at 40°C:

[0568] F6: 80 mM histidine pH 7.0, 8.8% (w / v) trehalose, 0.1% (w / v) poloxamer 188;

[0569] F7: 80 mM histidine + disodium hydrogen phosphate pH 7.5, 8.8% (w / v) trehalose, 0.1% (w / v) poloxamer 188;

[0570] F8: 20 mM sodium citrate pH 7.0, 8% (w / v) sucrose, 0.04% (w / v) polysorbate 80;

[0571] F9: 20 mM sodium citrate pH 7.0, 5% (w / v) mannitol, 0.04% (w / v) polysorbate 80.

[0572] Table 12. Excipient screening results

[0573] Experimental results

[0574] Appearance results showed that after the samples were placed under high temperature conditions at 40°C, a large number of visible particles appeared in the citric acid system at pH 7.0 (F8, F9) after one week, indicating poor stability of the preparations. However, no particles appeared in the histidine system at pH 7.0 and pH 7.5 (F6, F7) after four weeks of placement at 40°C, indicating that the preparations were relatively stable.

[0575] SEC results showed that after the samples were stored at 40°C for 4 weeks, the stability of the histidine system at pH 7.0 (F6, SEC monomer purity decreased by about 0.5%) was slightly better than that at pH 7.5 (F7, SEC monomer purity decreased by about 1.2%).

[0576] The NR-CE results showed that after being placed at 40°C for 4 weeks, the NR-CE main peak content of the sample in the histidine system pH 7.0 (F6) decreased by about 1.7% compared with T0; the NR-CE main peak content in the histidine system pH 7.5 (F7) decreased by about 4.4%, and the sample had good stability in the histidine system pH 7.0 (F6).

[0577] Example 12. Screening of antibacterial agents for fusion protein preparations

[0578] The following formulation containing 80 mM histidine, pH 7.0, and 100 mg / ml of the fusion protein was prepared and subjected to high temperature (40°C), repeated freeze-thaw cycles, and shaking stability tests.

[0579] F10: 0.5% (w / v) benzyl alcohol, 0.02% (w / v) polysorbate 80, 4.5% (w / v) sorbitol;

[0580] F11: 1% (w / v) benzyl alcohol, 4.5% (w / v) sorbitol;

[0581] F12: 1% (w / v) benzyl alcohol, 0.02% (w / v) polysorbate 80, 4.5% (w / v) sorbitol;

[0582] F13: 1% (w / v) benzyl alcohol, 0.04% (w / v) polysorbate 80, 4.5% (w / v) sorbitol;

[0583] F14: 1% (w / v) benzyl alcohol, 0.04% (w / v) polysorbate 80, 7% (w / v) sucrose;

[0584] F15: 0.25% (w / v) phenol, 0.02% (w / v) polysorbate 80, 4.5% (w / v) sorbitol;

[0585] F16: 0.25% (w / v) phenol, 0.04% (w / v) polysorbate 80, 4.5% (w / v) sorbitol;

[0586] Table 13. Antibacterial agent screening appearance

[0587] Table 14. SEC monomer purity of antibacterial agent screening

[0588] Table 15. Purity of main peak of NR-CE for screening of antibacterial agents

[0589] Table 16. Screening of antibacterial agents iCIEF

[0590] Experimental results

[0591] Appearance results showed that after 2 weeks at 40°C, the F11 formulation began to show a large number of flocculent particles, indicating poor stability. The F15 formulation also showed a large number of visible particles after 4 weeks at 40°C, indicating poor stability. The remaining formulations showed good stability.

[0592] The SEC results showed that the stability of each sample was good after 5 freeze-thaw cycles, 3 days of shaking, and 4 weeks at 40°C. The SEC monomer purity did not decrease significantly, and there was no significant difference between the formulations.

[0593] The NR-CE results showed that the stability of each sample was good after repeated freezing and thawing for 5 times, shaking for 3 days, and storage at 40°C for 4 weeks. The purity of the NR-CE main peak did not decrease significantly, and there was no significant difference between the formulations.

[0594] The iCIEF results showed that after the samples were frozen and thawed 5 times and shaken for 3 days, the stability of each formulation was good.

[0595] Example 13. Other optional formulations

[0596] In addition, the present invention also provides fusion protein pharmaceutical preparations with other formulations, including but not limited to:

[0597] (1) 100 mg / mL fusion protein, 80 mg / mL sucrose, 0.4 mg / mL polysorbate 80, and 80 mM histidine buffer pH 7.0;

[0598] (2) 100 mg / mL fusion protein, 70 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 80 mM histidine buffer pH 7.0;

[0599] (3) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, and 80 mM histidine buffer pH 7.0;

[0600] (4) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.4 mg / mL polysorbate 80, and 80 mM histidine buffer pH 7.0;

[0601] (5) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, 5 mg / mL benzyl alcohol, and 80 mM histidine buffer, pH 7.0;

[0602] (6) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.2 mg / mL polysorbate 80, 10 mg / mL benzyl alcohol, and 80 mM histidine buffer, pH 7.0;

[0603] (7) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.4 mg / mL polysorbate 80, 10 mg / mL benzyl alcohol, and 80 mM histidine buffer, pH 7.0;

[0604] (8) 100 mg / mL fusion protein, 45 mg / mL sorbitol, 0.4 mg / mL polysorbate 80, 2.5 mg / mL phenol, and 80 mM histidine buffer, pH 7.0;

[0605] (9) 100 mg / mL fusion protein, 70 mg / mL sucrose, 0.4 mg / mL polysorbate 80, 5 mg / mL benzyl alcohol, and 80 mM histidine buffer, pH 7.0;

[0606] (10) 100 mg / mL fusion protein, 70 mg / mL sucrose, 0.4 mg / mL polysorbate 80, 10 mg / mL benzyl alcohol, and 80 mM histidine buffer, pH 7.0;

[0607] (11) 100 mg / mL fusion protein, 70 mg / mL sucrose, 0.4 mg / mL polysorbate 80, 2.5 mg / mL phenol, and 80 mM histidine buffer, pH 7.0;

[0608] (12) 100 mg / mL fusion protein, 70 mg / mL sucrose, 0.4 mg / mL polysorbate 80, 5 mg / mL phenol and 80 mM histidine buffer pH 7.0.

[0609] The experimental results show that the fusion protein preparations of the above formulations all have good stability and can be used in the preparation of fusion protein drugs.

Claims

1. A pharmaceutical composition comprising: a) a fusion protein comprising an insulin analogue having the general formula from N-terminus to C-terminus: B1-L1-A1; in, B1 is an insulin B chain analog, which contains the following amino acid sequence: FVX1QHLCGX2HLVEALX3X4VCGERGFX5Y (SEQ ID NO: 1), wherein X1 is selected from N, G or K, X2 is selected from S or E, X3 is selected from Y, H or E, X4 is selected from H, R, L or E, X5 is selected from F or H; A1 is an insulin A chain analog, which contains the following amino acid sequence: GIVEQCCZ1SICSLZ2QLENYCZ3 (SEQ ID NO: 2), wherein Z1 is selected from T, H or E, Z2 is selected from Y, D, S or E, Z3 is selected from G or N; L1 is a linker, preferably a GS linker and / or a linker having at least 5 Gs, more preferably a linker having an amino acid sequence as shown in SEQ ID NO: 3 or 52; and b) a buffer, wherein the buffer is selected from one or more of histidine or a salt thereof, acetic acid or a salt thereof, succinic acid or a salt thereof, citric acid or a salt thereof, and phosphate, preferably selected from one or more of histidine or a salt thereof, acetic acid or a salt thereof, and phosphate, more preferably selected from one or more of histidine or a salt thereof and phosphate.

2. The pharmaceutical composition according to claim 1, wherein X1 is selected from N or K, X2 is selected from S or E, X3 is selected from Y or H, X4 is selected from L, and X5 is selected from H; Z1 is selected from T, H or E, Z2 is selected from E, and Z3 is selected from G or N.

3. The pharmaceutical composition according to claim 1 or 2, wherein: The insulin B chain analogue comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 31-36, The insulin A chain analog comprises or is selected from the amino acid sequence shown in any one of SEQ ID NOs: 37-42; Preferably, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 4-9.

4. The pharmaceutical composition according to claim 2 or 3, wherein the fusion protein further comprises C1, wherein the C1 is selected from at least one of the following: an Fc region of an immunoglobulin, HSA, and an HSA binding domain; Preferably, the C1 is selected from the Fc region of IgG; More preferably, the C1 is selected from the Fc region of human IgG1, IgG2, and IgG4.

5. The pharmaceutical composition according to claim 4, wherein The C1 is located at the N-terminus or the C-terminus of the insulin analog, preferably at the C-terminus of the insulin analog.

6. The pharmaceutical composition according to claim 4 or 5, wherein The C1 comprises or is an amino acid sequence shown in any one of SEQ ID NOs: 17, 18, 48-51, or an amino acid sequence having at least 90% identity therewith.

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the fusion protein further comprises a linker L2, and C1 forms a fusion protein with B1-L1-A1 through L2; Preferably, L2 is selected from any one of the following: m Q i ) n , (G m A i ) n , (G m Q) n , (G m A) n 、(PGPQ) s ,in, m is each independently selected from an integer of 1 to 10, n is independently selected from an integer of 1 to 10, i are each independently selected from 0-4; s is an integer selected from 1 to 10; More preferably, L2 comprises or is an amino acid sequence shown in any one of SEQ ID NOs: 10-16.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the fusion protein comprises or is an amino acid sequence as shown in any one of SEQ ID NOs: 19-27, or an amino acid sequence having at least 90% sequence identity therewith.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the fusion protein is a dimer, preferably a homodimer.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein The insulin analog is an insulin receptor agonist having insulin receptor agonist activity.

11. The pharmaceutical composition according to any one of claims 1 to 10, further comprising a surfactant, preferably the surfactant is selected from one or more of polysorbate and poloxamer, more preferably the surfactant is polysorbate 80 or poloxamer 188.

12. The pharmaceutical composition according to any one of claims 1 to 11, further comprising one or more of a sugar and a polyol, preferably the sugar is selected from one or more of sucrose and trehalose, and the polyol is selected from one or more of mannitol, sorbitol and glycerol, more preferably the polyol is sorbitol or glycerol, and the sugar is sucrose.

13. The pharmaceutical composition according to any one of claims 1 to 12, further comprising a bacteriostatic agent, preferably the bacteriostatic agent comprises one or more of benzyl alcohol, phenol, and m-cresol.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pH of the pharmaceutical composition is 5-9.5, preferably 6-9, more preferably 6.5-8.5, most preferably 6.5-7.

5.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the concentration of the fusion protein is 0.01 mg / mL-500 mg / mL, preferably 0.1 mg / mL-400 mg / mL, more preferably 0.5 mg / mL-200 mg / mL, most preferably 1 mg / mL-150 mg / mL.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the concentration of the buffer is 1 mM-200 mM, preferably 5 mM-150 mM, more preferably 10 mM-120 mM, most preferably 10 mM-100 mM.

17. The pharmaceutical composition of any one of claims 11 to 16, wherein the concentration of the surfactant is 0.01 mg / mL-5 mg / mL, preferably 0.05 mg / mL-3 mg / mL, more preferably 0.08 mg / mL-1.5 mg / mL, most preferably 0.1 mg / mL-1 mg / mL.

18. The pharmaceutical composition of any one of claims 12 to 17, wherein the concentration of the polyol or sugar is 1 mg / mL-200 mg / mL, preferably 10 mg / mL-150 mg / mL, more preferably 30 mg / mL-120 mg / mL, most preferably 40 mg / mL-100 mg / mL.

19. The pharmaceutical composition of any one of claims 13 to 18, wherein the concentration of the bacteriostatic agent is 0.01 mg / mL-50 mg / mL, preferably 0.05 mg / mL-40 mg / mL, more preferably 0.1 mg / mL-30 mg / mL, most preferably 0.5 mg / mL-20 mg / mL.

20. A pharmaceutical composition comprising: The fusion protein according to any one of claims 1 to 10; A buffer selected from one or more of histidine or its salts and phosphates, preferably selected from one or more of histidine and disodium hydrogen phosphate; polysorbates or poloxamers; Sucrose, sorbitol or glycerin; Optionally, the composition further comprises a bacteriostatic agent, preferably the bacteriostatic agent comprises one or more of benzyl alcohol, phenol and m-cresol.

21. The pharmaceutical composition according to claim 20, comprising any one of the following 1)-4): 1) 0.01 mg / mL-500 mg / mL of the fusion protein according to any one of claims 1 to 10; 1mM-200mM buffer, which is selected from one or more of histidine or its salts, phosphates, preferably selected from one or more of histidine and disodium hydrogen phosphate; 0.01mg / mL-5mg / mL polysorbate or poloxamer; 1mg / mL-200mg / mL sucrose or sorbitol; Optionally, the pharmaceutical composition further comprises 0.01 mg / mL-50 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises one or more of benzyl alcohol, phenol and m-cresol; And the pH of the pharmaceutical composition is 5-9.5; 2) 0.1 mg / mL-400 mg / mL of the fusion protein according to any one of claims 1 to 10; 5mM-150mM buffer, which is selected from one or more of histidine or its salts, phosphates, preferably selected from one or more of histidine and disodium hydrogen phosphate; 0.05mg / mL-3mg / mL polysorbate or poloxamer; 10mg / mL-150mg / mL sucrose or sorbitol; Optionally, the pharmaceutical composition further comprises 0.05 mg / mL-40 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises one or more of benzyl alcohol, phenol and m-cresol; And the pH of the pharmaceutical composition is 6-9; 3) 0.5 mg / mL-200 mg / mL of the fusion protein according to any one of claims 1 to 10; 10mM-120mM buffer, which is selected from one or more of histidine or its salts, phosphates, preferably selected from one or more of histidine and disodium hydrogen phosphate; 0.08mg / mL-1.5mg / mL polysorbate or poloxamer; 30mg / mL-120mg / mL sucrose or sorbitol; Optionally, the pharmaceutical composition further comprises 0.1 mg / mL-30 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises one or more of benzyl alcohol, phenol and m-cresol; And the pH of the pharmaceutical composition is 6.5-8.5; 4) 1 mg / mL-150 mg / mL of the fusion protein according to any one of claims 1 to 10; 10mM-100mM buffer, which is selected from one or more of histidine or its salts, phosphates, preferably selected from one or more of histidine and disodium hydrogen phosphate; 0.1mg / mL-1mg / mL polysorbate or poloxamer; 40mg / mL-100mg / mL sucrose or sorbitol; Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises one or more of benzyl alcohol, phenol and m-cresol; And the pH of the pharmaceutical composition is 6.5-7.

5.

22. The pharmaceutical composition according to claim 21, comprising any one of the following 1)-13): 1) 1-200 mg / mL of the fusion protein according to any one of claims 1 to 10; about 80 mM or about 40 mM histidine; 0.1mg / mL-1mg / mL polysorbate 80 or poloxamer 188; about 70 mg / mL sucrose, about 60 mg / mL sucrose, or about 45 mg / mL sorbitol; And the pH of the pharmaceutical composition is 6.5-7.5; Optionally, the pharmaceutical composition further comprises 0.5 mg / mL-20 mg / mL of an antibacterial agent, wherein the antibacterial agent comprises one or more of benzyl alcohol, phenol and m-cresol; 2) 1-200 mg / mL of the fusion protein according to any one of claims 1 to 10, preferably about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL or 150 mg / mL; About 80 mM histidine; about 0.2 mg / mL or about 0.4 mg / mL polysorbate 80; about 70 mg / mL sucrose or about 45 mg / mL sorbitol; and the pH of the pharmaceutical composition is about 6.5, about 7.0, or about 7.5; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 3) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.2 mg / mL polysorbate 80; About 70 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.0; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 4) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.4 mg / mL polysorbate 80; About 70 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.0; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 5) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.2 mg / mL polysorbate 80; About 45 mg / mL sorbitol; and the pH of the pharmaceutical composition is about 7.0; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 6) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.4 mg / mL polysorbate 80; About 45 mg / mL sorbitol; and the pH of the pharmaceutical composition is about 7.0; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent, wherein the bacteriostatic agent comprises about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 7) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.2 mg / mL polysorbate 80; About 70 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.5; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 8) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.4 mg / mL polysorbate 80; About 70 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.5; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 9) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.2 mg / mL polysorbate 80; About 45 mg / mL sorbitol; and the pH of the pharmaceutical composition is about 7.5; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 10) about 100 mg / mL of the fusion protein of any one of claims 1 to 10; About 80 mM histidine; About 0.4 mg / mL polysorbate 80; About 45 mg / mL sorbitol; and the pH of the pharmaceutical composition is about 7.5; Optionally, the pharmaceutical composition further comprises a bacteriostatic agent comprising about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 11) 1-200 mg / mL of the fusion protein of any one of claims 1 to 10, preferably about 1 mg / mL, about 20 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 80 mg / mL, about 100 mg / mL, about 120 mg / mL or 150 mg / mL; About 40 mM histidine; About 0.4 mg / mL Poloxamer 188; About 60 mg / mL sucrose; and the pH of the pharmaceutical composition is about 6.5, about 7.0, or about 7.5; Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol, and m-cresol, preferably about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 12) about 40 mg / mL or about 60 mg / mL of the fusion protein of any one of claims 1 to 10; About 40 mM histidine; About 0.4 mg / mL Poloxamer 188; About 60 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.0; Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol, and m-cresol, preferably about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol; 13) about 40 mg / mL or about 60 mg / mL of the fusion protein of any one of claims 1 to 10; About 40 mM histidine; About 0.4 mg / mL Poloxamer 188; About 60 mg / mL sucrose; and the pH of the pharmaceutical composition is about 7.5; Optionally, the pharmaceutical composition further comprises one or more of benzyl alcohol, phenol and m-cresol, preferably about 5 mg / mL or about 10 mg / mL benzyl alcohol, about 2.5 mg / mL or about 5 mg / mL phenol, or about 1 mg / mL or about 3 mg / mL m-cresol.

23. A lyophilized preparation, which can form the pharmaceutical composition of any one of claims 1 to 22 after being reconstituted, or which is obtained by freeze-drying the pharmaceutical composition of any one of claims 1 to 22.

24. A reconstituted solution, wherein the reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 23.

25. A product comprising a container containing the pharmaceutical composition of any one of claims 1 to 22, the lyophilized preparation of claim 23, or the reconstituted solution of claim 24.

26. Use of the pharmaceutical composition according to any one of claims 1 to 22, the lyophilized preparation according to claim 23 or the reconstituted solution according to claim 24 in the preparation of a drug for treating diabetes and its complications or lowering blood sugar; Preferably, the diabetes and its complications are selected from: type I diabetes, type II diabetes and its complications; More preferably, the complication is selected from diabetes-related heart disease, stroke, retinopathy, neuropathy or nephropathy.

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