Growth hormone F C Fusion protein injection and use thereof

Abstract

The present application relates to a composition containing growth hormone fusion protein and its use. The composition comprises: growth hormone fusion protein, and at least one of buffer and protective agent; the use of the above composition in the preparation of a medicament for treating and / or preventing diseases related to growth hormone abnormalities. The composition of the present application can reduce or inhibit the generation of molecular fragments, improve the stability of growth hormone fusion protein during preparation and storage; and the composition does not require complex preparation process, can be directly prepared into injection with high stability, and has good long-acting pharmacokinetic characteristics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a growth hormone F C The present application belongs to the field of biological medicine, and particularly relates to a growth hormone F BACKGROUND

[0002] Growth hormone (GH) is an endogenous hormone secreted by the anterior pituitary, which plays an important role in maintaining human growth and metabolism. Human growth hormone (hGH) is widely used to treat short stature caused by GH deficiency (GHD) or insufficiency and other growth disorders. Due to the short half-life of GH, daily subcutaneous injection is required, which greatly increases the pain of the treated person. In order to prolong the half-life, long-acting protein drugs (such as fusion protein drugs, PEGylated protein drugs) have become the mainstream of current biotechnology drug research and development.

[0003] The common long-acting growth hormone drugs at present mainly include growth hormone fusion protein drugs and PEGylated growth hormone protein drugs, which are usually in the form of lyophilized powder to maintain the stability of long-acting protein drugs. The dosage form of the growth hormone fusion protein drug developed in the clinical trial disclosed is also mainly lyophilized powder. However, when using lyophilized powder for injection, the injection steps are more, and repeated extraction is required, which increases the risk of glass debris particles and microorganisms. The injection solution of long-acting growth hormone drugs does not need to be repeatedly extracted, and has high safety.

[0004] Long-acting growth hormone drugs are generally administered once every 7 days or even 14 days by subcutaneous injection. Administration once every 7-14 days means a significant increase in the single dose, and in addition, the subcutaneous administration volume of children is usually limited to less than 1 ml, so long-acting growth hormone preparations need to be prepared into a higher concentration. The solubility of Fc fusion protein is generally low, so it is technically challenging to prepare a long-acting growth hormone injection solution with high concentration and good stability.

[0005] Therefore, it is urgent to develop a long-acting growth hormone drug injection solution with high concentration and good stability. SUMMARY

[0006] The present application aims to at least partially solve one of the problems in the prior art. To this end, the present application provides a composition containing a growth hormone fusion protein, which can reduce or inhibit the generation of molecular fragments, improve the stability of the growth hormone fusion protein in the composition, and particularly can be prepared into an injection solution with high stability.

[0007] The present application is based on the following findings of the inventors:

[0008] The long-acting growth hormone protein drugs commonly used at present mainly include growth hormone fusion protein drugs and PEGylated growth hormone protein drugs, etc. They are usually in the form of lyophilized powder to maintain the stability of the long-acting protein drugs. For example, patent CN101374536A discloses a drug in which PEG is covalently coupled to growth hormone. The drug is finally in the form of lyophilized powder to maintain the stability of the PEGylated growth hormone protein; patent CN108498467A discloses a lyophilized powder injection of recombinant growth hormone, which uses a formulation of protective agents, excipients, and surfactants, etc. The excipient replaces water and binds to the protein during lyophilization, thereby providing a skeleton support. Meanwhile, a surfactant is added to prevent particle aggregation and form a hydrophilic structure around the molecules.

[0009] However, the lyophilized powder is currently prepared by freeze-drying technology. For the lyophilized powder of long-acting growth hormone protein drugs, the original spatial structure of the growth hormone protein is changed during the preparation process, and the polymer is increased. Long-term use is prone to produce antibodies. Moreover, the injection steps of the lyophilized powder are more, and repeated extraction is required, which increases the risk of glass debris particles and microorganisms in the lyophilized powder injection.

[0010] The injection solution of long-acting growth hormone protein drugs has a simple preparation method, does not need to be dried during the preparation process, does not change the original spatial structure of the growth hormone protein, and does not need to be repeatedly extracted during injection, thereby being safe. The chemical degradation of short-acting growth hormone, i.e., growth hormone protein molecules not connected with PEG or Fc, is usually reported to be the loss or addition of part of groups such as deamidation and oxidation. The inventors found that growth hormone Fc fusion protein is prone to molecular chain segment truncation. The truncation position can be the growth hormone fragment or the linker or disulfide bond at the Fc end. Fc is an indispensable part of growth hormone Fc fusion protein to achieve the prolongation of metabolic half-life in vivo and realize long-acting. Therefore, the generation of molecular fragments (molecular truncation) during the preparation or storage of growth hormone fusion protein will affect the long-acting pharmacokinetic characteristics (referred to as long-acting pharmacokinetic characteristics).

[0011] Therefore, in one aspect of the present application, a composition is provided. According to an embodiment of the present application, the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protective agent. The composition of the present application can reduce or inhibit the generation of molecular fragments, improve the stability of the growth hormone fusion protein during preparation and storage.

[0012] In another aspect of the present application, the present application provides an injection solution. According to an embodiment of the present application, the injection solution comprises: a growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and the histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg / mL, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application can slow down or inhibit the generation of molecular fragments, and has the advantage of high stability.

[0013] In another aspect of the present application, the present application provides an injection solution. According to an embodiment of the present application, the injection solution comprises: a growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and the histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg / mL, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application can slow down or inhibit the generation of molecular fragments, and has the advantage of high stability.

[0014] In another aspect of the present application, the present application provides an injection solution. According to an embodiment of the present application, the injection solution comprises: a growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and the histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg / mL, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application can slow down or inhibit the generation of molecular fragments, and has the advantage of high stability.

[0015] In another aspect of the present application, the present application provides use of the aforementioned composition or the aforementioned injection solution in preparation of a medicament for treating and / or preventing a disease associated with abnormal growth hormone.

[0016] In another aspect of the present application, the present application provides an injection device or container. According to an embodiment of the present application, the injection device or container comprises the aforementioned composition or the aforementioned injection solution. The injection device or container of the present application facilitates storage of the aforementioned composition or the aforementioned injection solution.

[0017] Additional aspects and advantages of the present application will be in part apparent and in part pointed out below in the description of embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description of embodiments of the present application, taken in conjunction with the accompanying drawings in which:

[0019] Figure 1 Results of detection of the %HMW of the aggregates by SEC-HPLC in Example 1 of the present application;

[0020] Figure 2 Results of detection of the %LMW of the truncated fragments by CE-SDS in Example 1 of the present application;

[0021] Figure 3 Results of detection of the %HMW of the aggregates by SEC-HPLC in Example 2 of the present application;

[0022] Figure 4 Results of detection of the %LMW of the truncated fragments by CE-SDS in Example 2 of the present application;

[0023] Figure 5 Results of detection of the %HMW of the aggregates by SEC-HPLC in Example 2 of the present application;

[0024] Figure 6 Results of detection of the %LMW of the truncated fragments by CE-SDS in Example 2 of the present application;

[0025] Figure 7 Results of detection of the %HMW of the aggregates by SEC-HPLC in Example 4 of the present application;

[0026] Figure 8 Results of detection of the %LMW of the truncated fragments by CE-SDS in Example 4 of the present application. DETAILED DESCRIPTION

[0027] Embodiments of the present application are described in detail below. The embodiments described below are exemplary in nature and are intended to explain the present application, and should not be considered limiting the present application.

[0028] It should be noted that the terms "first", "second" are used only for descriptive purposes, and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise stated.

[0029] The endpoints of the ranges and any values described herein are not limited to the precise values stated. The ranges and values should be construed to be roughly about the ranges or values. For ranges, the endpoints are included as well as intervening points within the range. For values, the value includes the stated value as well as approximately the value.

[0030] In this document, the terms "comprises" or "comprising" are open-ended, that is, they mean including, but not limited to, the indicated features.

[0031] In this document, the terms "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs, and instances where it does not.

[0032] In this document, the term "growth hormone abnormality related disease" generally means a disease caused by abnormality of growth hormone, for example, a disease related to growth hormone deficiency or dysregulation, including but not limited to, pediatric growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner's Syndrome, Prader Willi Syndrome, intrauterine growth retardation, idiopathic short stature, renal failure, dysregulation during chemotherapy treatment and AIDS treatment. Growth hormone deficiency can include congenital or acquired deficiency. With regard to congenital deficiency, growth hormone deficiency can occur when the pituitary does not develop a disorder in growth hormone secretion. Acquired growth hormone deficiency can occur due to hypoxia resulting from brain tissue damage due to difficulty in delivery. Other causes of growth hormone deficiency include pituitary damage caused by radiation for treatment of brain tumors or postnatal tuberculous meningitis. Growth hormone deficiency exhibits symptoms such as growth retardation and short stature, and congenital growth hormone deficiency exhibits symptoms of hypoglycemia from the neonatal period. In addition, children exhibit symptoms such as increased anxiety and decreased energy.

[0033] The term "treatment" as used herein means for the purposes of obtaining a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce the symptoms of, or inhibit the disease in the individual, including, but not limited to, the administration of a drug comprising a compound described herein to an individual in need thereof.

[0034] In the context of the present application, the terms "identity," "homology," or "similarity" are used in the context of describing an amino acid sequence or nucleic acid sequence relative to a reference sequence, and are determined by the percent of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, for example, see, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to determine identity, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In the context of the present application, the term "at least 90% identity" means at least 90%, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to a reference sequence.

[0036] In this document, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus), and / or has an internal deletion. For example, an Fc fragment may include a hinge region, a CH2 region, and a CH3 region, or a portion thereof, such as containing only the CH2 and CH3 regions; a serum albumin fragment may be serum albumin or a truncated fragment of serum albumin; a growth hormone fragment may be growth hormone or a truncated fragment of growth hormone.

[0037] This invention proposes a composition and an injection solution, as well as their preparation method and uses, which will be described in detail below.

[0038] Composition

[0039] In one aspect of the invention, a composition is provided. According to embodiments of the invention, the composition comprises: a growth hormone fusion protein, and at least one of a buffer and a protectant. The inventors have discovered through extensive experimentation that the addition of a buffer and / or protectant can reduce or inhibit the generation of molecular fragments, thereby improving the stability of the growth hormone fusion protein during preparation and storage.

[0040] It should be noted that the "growth hormone fusion protein" mentioned in this invention generally refers to a protein obtained by fusing a growth hormone fragment with the amino acid sequence of natural growth hormone with other protein fragments or polypeptide fragments. Specifically, the N-terminus of the growth hormone fragment is linked to the C-terminus of the protein fragment or polypeptide fragment, or vice versa. Furthermore, the growth hormone fragment and the protein fragment or polypeptide fragment can be linked by a linker peptide. Preferably, the protein fragment or polypeptide fragment is a fragment with a specific function, such as enhancing the binding activity of the growth hormone fragment to the growth hormone receptor or reducing the ADCC effect of growth hormone. The protein fragment or polypeptide fragment includes, but is not limited to, Fc fragments and serum albumin fragments.

[0041] According to an embodiment of the present invention, the growth hormone fusion protein is growth hormone F. C Fusion protein. The inventors unexpectedly discovered through experimentation that the composition of this invention has an effect on growth hormone F... C Fusion proteins have a good protective effect. During the production and / or storage of growth hormone Fc fusion proteins, they can slow down or inhibit the breakage of growth hormone and Fc fragments, as well as the further chemical degradation of growth hormone and Fc after breakage, thereby reducing or avoiding the generation of molecular fragments and preventing excessive molecular fragments from affecting its long-acting pharmacokinetic characteristics.

[0042] It should be noted that the "growth hormone F" mentioned in this invention... CThe "fusion protein" generally refers to a protein fused by a growth hormone fragment and an Fc fragment C The Fc fragment can be derived from a murine antibody, a human antibody, and a primate antibody or a mutant thereof. The Fc fragment refers to the Fc end of an immunoglobulin IgG, which has different subtypes, such as IgG1, IgG2, IgG3, and IgG4. The Fc fragment can be a complete Fc fragment of a natural murine, human, or primate antibody (IgG), or a complete Fc fragment of IgG with some amino acids inserted, replaced, or deleted by point mutation of amino acids.

[0043] Exemplarily, the growth hormone Fc fusion protein of the present application is the growth hormone Fc fusion protein mentioned in patent application numbers 202210459224.3, 202210395423.2, and 202210395418.1. C The fusion protein can be the growth hormone Fc fusion protein mentioned in patent application numbers 202210459224.3, 202210395423.2, and 202210395418.1. C The fusion protein.

[0044] According to an embodiment of the present application, the buffer includes at least one selected from the group consisting of histidine and histidine hydrochloride, sodium citrate and citric acid, and acetic acid / acetic acid sodium. Thus, the stability of the growth hormone fusion protein can be improved.

[0045] According to an embodiment of the present application, the buffer includes histidine and histidine hydrochloride. The inventors have found through experiments that, compared with other buffer pairs (such as sodium citrate / citric acid and acetic acid / acetic acid sodium), the buffer pair of histidine and histidine hydrochloride can effectively reduce or avoid the generation of molecular fragments in the composition, improve the stability of the composition, and prevent the excessive molecular fragments from affecting the long-acting pharmacokinetic characteristics of the composition.

[0046] It should be noted that, in this document, the molar concentration of the buffer is the total molar concentration of the buffer pair. Exemplarily, the molar concentration of the buffer is the total molar concentration of histidine and histidine hydrochloride. The "histidine and histidine hydrochloride" is synonymous with the "histidine and histidine hydrochloride buffer pair".

[0047] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein and the buffer is (20-90) mg:(0.005-0.04) mmol, preferably (50-70) mg:(0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein can be further improved.

[0048] According to an embodiment of the present application, the protective agent includes at least one selected from the group consisting of sucrose, trehalose, sorbitol, and mannitol. Thus, the osmotic pressure in the composition can be adjusted, and the stability of the growth hormone fusion protein in the composition can be further improved.

[0049] According to an embodiment of the present application, the molar ratio of the growth hormone fusion protein and the protective agent is (20-90) mg : (20-90) mg, preferably (50-70) mg : (40-70) mg, and more preferably (50-70) mg : (42-64) mg. Thus, the stability of the growth hormone fusion protein can be further improved. According to an embodiment of the present application, the protective agent is preferably sucrose.

[0050] According to an embodiment of the present application, the protective agent includes sucrose and trehalose. The inventors have found through experiments that the combination of sucrose and trehalose prevents protein aggregation and further improves the stability of the growth hormone fusion protein.

[0051] According to an embodiment of the present application, the mass ratio of the growth hormone fusion protein, sucrose, and trehalose is (20-90) mg : (20-45) mg : (20-45) mg, preferably (40-90) mg : (15-35) mg : (15-35) mg. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.

[0052] According to an embodiment of the present application, the composition includes the growth hormone fusion protein, a buffer, and a protective agent. Thus, the stability of the growth hormone fusion protein can be further improved.

[0053] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the protective agent, and the buffer is (20-90) mg : (20-90) mg : (0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.

[0054] According to an embodiment of the present application, the composition further includes at least one of a stabilizer, a surfactant, an antioxidant, and a metal chelating agent. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.

[0055] According to an embodiment of the present application, the stabilizer includes at least one selected from the group consisting of tromethamine and tromethamine hydrochloride. The inventors have found that tromethamine or tromethamine hydrochloride forms a non-covalent bond, such as a hydrogen bond, with a growth hormone molecule (e.g., an hGH molecule) to increase the reaction energy, thereby making the growth hormone fusion protein more stable and further improving the stability of the growth hormone fusion protein.

[0056] According to an embodiment of the present application, the stabilizer is tromethamine. Thus, the stability of the growth hormone fusion protein can be further improved.

[0057] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein and the stabilizer is (20-90) mg:(0.01-0.05) mmol, preferably (50-70) mg:(0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein can be further improved.

[0058] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein and the antioxidant is (20-90) mg:(0.01-0.02) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.

[0059] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein and the metal chelating agent is (20-90) mg:(0.00015-0.01) mmol, preferably (40-90) mg:(0.00015-0.0003) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.

[0060] According to an embodiment of the present application, the antioxidant includes methionine. Thus, the antioxidant can competitively bind to dissolved oxygen in the injection solution, protecting the growth hormone fusion protein from oxidation.

[0061] According to an embodiment of the present application, the metal chelating agent includes edetate disodium (also known as EDTA-2Na). Thus, the metal chelating agent can form a chelate with metal ions, preventing the metal ions from catalyzing protein degradation.

[0062] According to an embodiment of the present application, the mass ratio of the growth hormone fusion protein and the surfactant is (1-50):(0.1-1).

[0063] According to an embodiment of the present application, the surfactant includes at least one selected from the group consisting of Tween 80, Tween 20, and poloxamer P188.

[0064] According to an embodiment of the present application, the composition includes the growth hormone fusion protein, a buffer, and a stabilizer. Thus, the stability of the growth hormone fusion protein can be further improved.

[0065] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the buffer, and the stabilizer is (20-90) mg:(0.015-0.03) mmol:(0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.

[0066] In a preferred embodiment of the present application, the buffer comprises histidine and histidine hydrochloride, and the stabilizer is tromethamine. The inventors have found through experiments that when the composition contains both the histidine and histidine hydrochloride buffer pair and tromethamine, the breakage between the growth hormone and other protein fragments or polypeptide fragments, and the further chemical degradation of the growth hormone and protein fragments or polypeptide fragments after breakage can be further slowed down or inhibited, the generation of molecular fragments in the composition is reduced, and the protection effect on the growth hormone fusion protein is improved.

[0067] According to an embodiment of the present application, the composition comprises the growth hormone fusion protein, the protective agent, and the stabilizer. Thus, the stability of the growth hormone fusion protein can be further improved.

[0068] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the protective agent, and the stabilizer is (20-90) mg:(40-90) mg:(0.015-0.035) mmol, preferably (40-90) mg:(50-80) mg:(0.015-0.035) mmol, and more preferably (40-90) mg:(51-77) mg:(0.015-0.035) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.

[0069] In a preferred embodiment of the present application, the stabilizer is tromethamine, and the protective agent comprises sucrose. Thus, the stability of the growth hormone fusion protein can be further improved.

[0070] In a preferred embodiment of the present application, the stabilizer is tromethamine, and the protective agent comprises sucrose and trehalose. Thus, the stability of the growth hormone fusion protein can be further improved.

[0071] According to an embodiment of the present application, the composition comprises the growth hormone fusion protein, the buffer, the protective agent, and the stabilizer. The inventors have found through a large number of experiments that the addition of the buffer, the protective agent, and the stabilizer can effectively protect the growth hormone fusion protein, slow down or inhibit the breakage between the growth hormone fragments and other protein fragments or polypeptide fragments, and the further chemical degradation of the growth hormone fragments and protein fragments or polypeptide fragments after breakage, thereby reducing the generation of molecular fragments in the composition and improving the stability of the composition. Moreover, the inventors have also found through a large number of experiments that due to the high stability of the growth hormone fusion protein in the composition, the composition does not need a complex preparation process and can be directly prepared into an injection with high stability, and the injection has good long-acting pharmacokinetic characteristics.

[0072] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (40-90) mg : (20-90) mg : (0.01-0.05) mmol : (0.005-0.04) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be improved.

[0073] According to an embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the protective agent, the stabilizer and the buffer is (50-70) mg : (30-70) mg : (0.015-0.035) mmol : (0.015-0.03) mmol. Thus, the stability of the growth hormone fusion protein in the composition can be further improved.

[0074] In a preferred embodiment of the present application, the mass molar ratio of the growth hormone fusion protein, the tromethamine, the histidine and the histidine hydrochloride buffer pair, the sucrose and the trehalose is (50-70) mg : (0.015-0.035) mmol : (0.015-0.03) mmol : (20-45) mg : (20-45) mg. Thus, the breaking between the growth hormone and other protein fragments or polypeptide fragments, and the chemical degradation of the growth hormone and other protein fragments or polypeptide fragments after breaking can be slowed down or inhibited, the production of molecular fragments in the injection solution is reduced, and the composition has the advantages of low molecular fragment content and high stability. In particular, the Nr-CE SDS and SEC purity of the composition under different storage conditions is 90% or more.

[0075] According to an embodiment of the present application, the composition is an injection solution.

[0076] According to an embodiment of the present application, the pH value of the composition is 6.1-7.2, preferably 6.4-7.2.

[0077] According to an embodiment of the present application, the concentration of the growth hormone fusion protein is 20-90 mg / mL, preferably 50-70 mg / mL.

[0078] According to an embodiment of the present application, the concentration of the protective agent is 20-90 mg / mL, preferably 30-70 mg / mL.

[0079] According to an embodiment of the present application, the concentration of the buffer is 5-40 mM, preferably 15-30 mM.

[0080] According to an embodiment of the present application, the concentration of the stabilizer is 10-50 mM, preferably 15-35 mM.

[0081] According to an embodiment of the present application, the concentration of the antioxidant is 10-20 mM.

[0082] According to embodiments of the present application, the concentration of the chelating agent is 0.15-1 mM, preferably 0.15-0.3 mM.

[0083] According to embodiments of the present application, the growth hormone F C The fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1-4 or an amino acid sequence having at least 90% identity thereto.

[0084] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFRNTGKGGEEKKKEKEEKENEEEEEREPECPESKYGPPCPPCPAPEVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 1).

[0085] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVYHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF (SEQ ID NO: 2)

[0086] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 3)

[0087] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGFGGGGSGGGGSGGGGSGGGGSGGGGSESKYGPPCPPCPAPPVEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 4)

[0088] Injection solution

[0089] In yet another aspect of the present application, the present application provides an injection solution. According to an embodiment of the present application, the injection solution comprises: a growth hormone fusion protein, histidine, hydrochloric acid histidine, tromethamine and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and the hydrochloric acid histidine is 15-30 mM, the concentration of the tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg / mL, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application can slow down or inhibit the cleavage between the growth hormone and other protein fragments or polypeptide fragments, and the chemical degradation of the growth hormone and other protein fragments or polypeptide fragments after cleavage during production and / or storage, reduce the generation of molecular fragments in the injection solution, and has the advantages of low molecular fragment content and high stability. Moreover, compared with other solutions, the injection solution can slow down the decrease of the Nr-CE SDS and SEC purity under room temperature illumination and 40°C pressure conditions, indicating that it will be more stable in the long-term stability of biological products under the conventional 2-8°C, dark storage conditions.

[0090] According to embodiments of the present application, the growth hormone fusion protein is growth hormone F C fusion protein, in particular growth hormone F C fusion protein, in particular growth hormone F C fusion protein.

[0091] In another aspect of the present application, an injection solution is provided. According to embodiments of the present application, the injection solution comprises a growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg / mL, and the concentration of the methionine is 10-20 mM, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application is further observed to have improved anti-aggregation.

[0092] According to embodiments of the present application, the growth hormone fusion protein is growth hormone F C fusion protein, in particular growth hormone F C fusion protein, in particular growth hormone F C fusion protein.

[0093] In another aspect of the present application, an injection solution is provided. According to embodiments of the present application, the injection solution comprises a growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine and a protective agent; the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of the histidine and histidine hydrochloride is 15-30 mM, the concentration of the tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg / mL, and the concentration of the methionine is 10-20 mM, based on the total volume of the injection solution; wherein the protective agent is sucrose, and the concentration of the sucrose is 40-90 mg / mL; or the protective agent is sucrose and trehalose, the concentration of the sucrose is 20-45 mg / mL, and the concentration of the trehalose is 20-45 mg / mL. The injection solution of the present application is further observed to have improved anti-aggregation.

[0094] According to embodiments of the present application, the growth hormone fusion protein is growth hormone F C fusion protein, in particular growth hormone F CThe growth hormone fusion protein is as described above in the composition C The growth hormone fusion protein.

[0095] Method for preparing the composition and injection

[0096] In another aspect of the present application, the present application provides a method for preparing the composition as described above. According to an embodiment of the present application, the method comprises: mixing the growth hormone fusion protein, the buffer, the protective agent and the stabilizer to obtain the composition; wherein the buffer comprises sodium citrate and citric acid. The method of the present application can prepare the composition, and the preparation method is simple.

[0097] According to an embodiment of the present application, the composition is an injection, and the mixing is performed by: performing a first mixing of a first solution containing the growth hormone fusion protein and a second solution containing the protective agent; performing a second mixing of the buffer and the stabilizer, and optionally an antioxidant and / or a metal chelator; performing a first ultrafiltration of the first mixing product, and performing a third mixing of the first ultrafiltration product and the second mixing product to obtain the composition.

[0098] According to an embodiment of the present application, the concentration of the growth hormone fusion protein in the first solution is 1-10 mg / mL.

[0099] According to an embodiment of the present application, before the first ultrafiltration, the first mixing product is subjected to a first concentration to obtain a first concentrated solution with a concentration of the growth hormone fusion protein of 10-20 mg / mL.

[0100] According to an embodiment of the present application, the concentration of the growth hormone fusion protein in the third mixing product is 10-20 mg / mL.

[0101] According to an embodiment of the present application, after the third mixing, the third mixing product is subjected to a second concentration to obtain a second concentrated solution with a concentration of the growth hormone fusion protein of greater than 50 mg / mL.

[0102] According to an embodiment of the present application, the method further comprises: after the second concentration, diluting the second concentrated solution to obtain a composition with a concentration of the growth hormone fusion protein of 40-90 mg / mL.

[0103] Use

[0104] In another aspect of the present application, the present application provides a use of the composition as described above, the injection as described above, or the composition prepared according to the method as described above in the preparation of a medicament for treating and / or preventing diseases related to growth hormone abnormalities.

[0105] According to embodiments of the present application, the growth hormone abnormality-related disease includes at least one selected from the group consisting of childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, a disease caused by a catabolic state during chemotherapy treatment and AIDS treatment, and intrauterine growth retardation.

[0106] Method of treating and / or preventing a growth hormone abnormality-related disease

[0107] In another aspect of the present application, the present application provides a method of preventing and / or treating a growth hormone abnormality-related disease. According to embodiments of the present application, the method includes administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection solution to a subject. According to embodiments of the present application, the method is effective in preventing or treating a growth hormone abnormality-related disease.

[0108] The effective amount of the composition or the injection solution of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art according to various factors (e.g., through clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the body weight of the patient, the immune status of the patient, the route of administration, etc. For example, several divided doses can be administered daily, or the dose can be proportionally reduced, as the acute condition requires.

[0109] According to embodiments of the present application, the route of administration of the method includes subcutaneous injection or intravenous injection.

[0110] According to embodiments of the present application, the growth hormone abnormality-related disease includes at least one selected from the group consisting of childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, Prader-Willi syndrome, renal failure, a disease caused by a catabolic state during chemotherapy treatment and AIDS treatment, and intrauterine growth retardation.

[0111] Injection device or container

[0112] In another aspect of the present application, the present application provides an injection device or container. According to embodiments of the present application, the injection device or container includes the aforementioned composition or the aforementioned injection solution. The injection device or container of the present application is convenient for storing the aforementioned composition or the aforementioned injection solution.

[0113] In this context, the injection device or container of the present application should be construed broadly and includes any product that can accommodate the composition or the injection solution of the present application.

[0114] According to an embodiment of the present application, the injection device or container comprises at least one selected from a group consisting of a vial and a syringe.

[0115] According to an embodiment of the present application, the syringe comprises at least one selected from a group consisting of an auto-injector and a pre-filled syringe.

[0116] The schemes of the present application will be explained below in connection with the examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be considered as limiting the scope of the present application. If no specific technique or condition is specified in the examples, the technique or condition described in the literature in the art or according to the product manual is used. If no manufacturer of the reagent or instrument is specified, it is a conventional product that can be commercially available.

[0117] The related protein of "high molecular weight" (HMW) species of the formulation mentioned herein refers to the content of any related protein eluted earlier than the main peak from a size exclusion chromatography column (SEC), and the content of HMW is used to indicate the degree of aggregation of the protein. %HMW refers to the percentage of the peak area of high molecular weight protein relative to the total sum of all detectable protein peaks in the formulation when detected by SEC-HPLC chromatography.

[0118] The related protein of "low molecular weight" (LMW) species mentioned herein refers to the content of any related protein eluted earlier than the main peak from a non-reducing capillary electrophoresis (nrCE-SDS detection). %HMW refers to the percentage of the peak area of high molecular weight protein relative to the total sum of all detectable protein peaks in the formulation when detected by SEC-HPLC chromatography, and the content of HMW is used to indicate the degree of degradation of active protein molecules to produce truncated fragments.

[0119] N / A in the examples herein refers to no addition.

[0120] It should be noted that the specific amino acid sequence of the human growth hormone Fc fusion protein used in the embodiments of the present application is as shown in SEQ ID NO: 1, which is prepared by a conventional method; and the remaining excipients are commercially available.

[0121] Example 1: Screening of buffer and pH value in human growth hormone Fc fusion protein injection

[0122] 1. The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the type and final concentration of each excipient are shown in Table 1.

[0123] 2. The preparation method of the injection is as follows:

[0124] 1) Take a certain volume of protein solution containing phosphate buffer solution, the concentration of human growth hormone Fc fusion protein in the protein solution is 2-3 mg / mL, and sucrose is added in an amount of 30 mg / mL. Then filter the particles with a 0.45 μm filter to obtain a filtrate.

[0125] 2) The filtrate is pre-concentrated using a milipore pellicon2 30KD ultrafiltration membrane package, and the concentration of human growth hormone Fc fusion protein is pre-concentrated to 10-20 mg / mL to obtain a protein pre-concentrate.

[0126] 3) According to the formulation in Table 1, the human growth hormone Fc fusion protein and other excipients except sucrose are formulated into a buffer solution, and the pH value of the buffer solution is adjusted to the specified value (see Table 1 for details) using hydrochloric acid or sodium hydroxide.

[0127] 4) The protein pre-concentrate is maintained at a fixed volume, and the phosphate buffer solution of the protein pre-concentrate is replaced with the buffer solution by ultrafiltration. Then continue to concentrate by ultrafiltration, and the target protein content is detected by Protein A-HPLC. The concentration of human growth hormone Fc fusion protein should be concentrated to 20 mg / ml or more, for example, about 20 mg / ml, 50 mg / ml, 70 mg / ml or 80 mg / ml, or up to about 90 mg / ml.

[0128] 5) A certain volume of buffer solution is added to the protein solution at the end of concentration to dilute to the intended concentration of human growth hormone Fc fusion protein in the preparation (see Table 1 for details), to obtain an injection stock solution. When the concentration of the concentrated human growth hormone Fc fusion protein is the same as the required concentration, no dilution treatment in step 4) is required.

[0129] 6) According to the storage conditions of the injection stock solution, after necessary thawing, mixing, and 0.2 μm sterilization filter filtration, the injection stock solution is divided into a Schlenk bottle, sealed, and the injection solution is obtained.

[0130] 3, then the injection solution obtained from each sample is subjected to SEC-HPLC and nrCE-SDS detection at 40°C, and the specific detection results are shown in Table 2 and Figures 1-2 .

[0131] The SEC-HPLC and nrCE-SDS detection is as follows:

[0132] SEC-HPLC separates sample molecules according to the pore size of the filler. Large molecules of the sample cannot enter or can only enter part of the pores, while smaller molecules can enter most or all of the pores, thereby separating proteins of different molecular weights. The main parameters of the SEC-HPLC detection method described herein are:

[0133] Parameter Parameter value Mode pH 7.0 100 mM phosphate buffer isocratic elution Chromatographic column Filler particle size 5 μm, column diameter * column length 7.8 * 30 mm Detection wavelength 280 nm Flow rate 0.5 mL / min Injection volume 50 μl Column temperature 30℃ Inj ector temperature 8℃

[0134] Nr-CE-SDS detection used AB SCIEX PA800plus capillary electrophoresis instrument and SDS gel buffer with the company's part number A30341, the main parameters of the equipment are as follows:

[0135]

[0136]

[0137] Table 1: The ratio of each component in the injection solution of samples 1-8

[0138]

[0139] Table 2: The detection results of SEC-HPLC and nrCE-SDS in samples 1-8

[0140]

[0141] From Tables 1-2 and Figures 1-2 It can be seen that in the injection solution of different buffer systems and pH conditions, the protein aggregate detected by SEC-HPLC (%HMW) and the truncated fragment content detected by CE-SDS (%LMW) are different. In terms of %HMW of SEC-HPLC, other systems such as citric acid system (citric acid / sodium citrate) and phosphate system (disodium hydrogen phosphate / sodium phosphate) increase with increasing pH, while histidine system (histidine / histidine hydrochloride) decreases with increasing pH, indicating that the histidine system can reduce aggregates when the pH in the range of 6.1-7.0 increases. In terms of %LMW of CE-SDS, the histidine system (histidine / histidine hydrochloride) has little change in the pH range of 6.1-7.0, while the citric acid system and the phosphate system increase with increasing pH, which will increase the truncated fragments. In summary, the results of histidine system are better than those of citric acid system and phosphate system in both SEC and CE. According to the trend of histidine system, it is also possible to try a higher pH, for example, pH 7.2.

[0142] Example 2: Screening of protective agents in human growth hormone Fc fusion protein injection

[0143] In order to screen the more suitable protective agent, the inventors tested the thermal stability and freeze-thaw protection of five protective agents on growth hormone fusion protein. The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection solution and the type and final concentration of each excipient are shown in Table 3, the preparation method and detection method of the injection solution are shown in Example 1, and the detection results are shown in Table 4 and Figures 3-4The results show that the order of advantages in thermal stability and anti-aggregation (HMW% of SEC) at high temperature and freeze-thaw is: trehalose ≈ sucrose > mannitol > arginine hydrochloride > sodium chloride. Arginine hydrochloride and sodium chloride belong to ionic salt protective agents, and the overall protective effect is not as good as other sugar alcohol non-ionic protective agents.

[0144] Table 3: The ratio of each component in the injection of samples 9-13

[0145]

[0146] Table 4: The detection results of SEC-HPLC and nrCE-SDS in samples 9-13

[0147]

[0148]

[0149] Further, the inventors tested the difference between non-ionic sugar alcohol protective agent combination and single protective agent. The final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the type and final concentration of each excipient are shown in Table 5. The preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Figures 5-6 In summary, the growth trend of CE-SDS and SEC for 30 days is evaluated, and sucrose and trehalose show a synergistic effect. The stability of sucrose and trehalose combination is better than that of single use.

[0150] Table 5: The ratio of each component in the injection of samples 14-18

[0151]

[0152] Example 3: Screening of stabilizers in human growth hormone Fc fusion protein injection

[0153] Stabilizers are components that are beneficial to the stability of active molecules. For example, some stabilizers can non-covalently bind to active molecules through molecular interactions (such as hydrogen bonds, hydrophobic interactions), etc., to increase the energy required for their reactions. In order to screen the most suitable stabilizer, the inventors tested the thermal stability of growth hormone fusion protein with stabilizers, and found that Tris or Tris-hydrochloride (trisaminobutanol) has a similar effect. Tris has multiple hydrogen bond donors and acceptors, which can bind to protein molecules.

[0154] In this embodiment, the final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection solution and the type and final concentration of each excipient are shown in Table 6. The preparation method and detection method of the injection solution are shown in Example 1, and the detection results are shown in Table 7. Due to the adjustment of the upstream fermentation and purification process, the stability of the provided protein is worse, and significant aggregation and molecular truncation occur at 40°C, light, and 5°C. However, the results of the parallel comparison of the samples in Table 6 show that compared with the sample without the addition of a stabilizer, the addition of Tris improves the %LMW detected by CE-SDS by 1-2 percentage points under light and 5°C. Although it is not very significant, for the human growth hormone Fc fusion protein which is prone to LMW, this subtle improvement also has extraordinary value. Furthermore, the inventors further explored the amount of the added stabilizer, and the amount of Tris increased from 15-25 mM, and the improvement of the %LMW detected by CE-SDS increased accordingly.

[0155] Table 6: Proportion of each component in the injection solution of samples 19-23

[0156]

[0157] Table 7: Detection results of SEC-HPLC and nrCE-SDS in samples 19-23

[0158]

[0159]

[0160] Example 4: Screening of antioxidants and metal chelating agents in human growth hormone Fc fusion protein injection solution

[0161] In order to further improve the stability of the injection solution, the inventors screened antioxidants (methionine) and metal chelating agents (EDTA-2Na, also known as disodium edetate). The inventors found that methionine and disodium edetate have a certain stabilizing effect on the fusion protein, and the stability can be further improved by combining with stabilizers.

[0162] In this embodiment, the final concentration of human growth hormone Fc fusion protein (referred to as fusion protein) in the injection solution and the type and final concentration of each excipient are shown in Table 8. The preparation method and detection method of the injection solution are shown in Example 1, and the detection results are shown in Figures 7-8 and Table 9. The results show that compared with the sample without the addition of methionine and EDTA-2Na, the addition of methionine slightly improves the HMW, and the addition of 0.15-0.3 mM of EDTA-2Na can significantly improve the %LMW detected by CE-SDS, and also improve the aggregation (HMW) at high temperature of 40°C.

[0163] Table 8: The ratio of each component in the injection solution of samples 24-28

[0164]

[0165] Table 9: The detection results of SEC-HPLC and nrCE-SDS in samples 24, 26 and 28

[0166]

[0167] Example 5: Screening of the amount of histidine buffer added in the human growth hormone Fc fusion protein injection

[0168] The final concentration of the human growth hormone Fc fusion protein (referred to as fusion protein) in the injection and the type and final concentration of each excipient are shown in Table 10. The preparation method and detection method of the injection are shown in Example 1, and the detection results are shown in Table 11.

[0169] Table 10: The ratio of each component in the injection solution of samples 29-32

[0170]

[0171] Table 11: The detection results of SEC-HPLC and nrCE-SDS in samples 29-32

[0172]

[0173] As shown in Tables 10-11, compared with the 40 mM histidine system, the %LMW of CE-SDS in the 10-30 mM histidine system is lower. Therefore, the stability of the 10-30 mM histidine system is relatively better. Finally, under the joint action of appropriate ionic strength and corresponding protective agents and stabilizers, the sample preparation of the present embodiment has accumulated stability data for 6 months under real shelf life storage conditions at 2-8°C. From the SEC and CE-SDS results, the growth is slow, and it is expected that the SEC and CE-SDS purity can be maintained at more than 90% within the shelf life of 18-24 months.

[0174] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples without contradiction.

[0175] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and that variations, modifications, substitutions and changes can be made by those skilled in the art without departing from the scope of the present application.

Claims

1. A composition, characterized in that, include: Growth hormone fusion protein, and buffers and protectants; and at least one of stabilizers, surfactants, antioxidants and metal chelators; The buffer includes histidine and histidine hydrochloride, and the protectant includes at least one selected from sucrose and trehalose; The mass and molar ratio of the growth hormone fusion protein, protectant, and buffer is (50–70) mg : (40–90) mg : (0.015–0.03) mmol. The growth hormone fusion protein is growth hormone F. C Fusion protein.

2. The composition according to claim 1, characterized in that, The protective agents include sucrose and trehalose.

3. The composition according to claim 1, characterized in that, The mass ratio of the growth hormone fusion protein, sucrose and trehalose is (20~90) mg: (20~45) mg: (20~45) mg.

4. The composition according to claim 1, characterized in that, The mass ratio of the growth hormone fusion protein, sucrose and trehalose is (50~70) mg: (15~35) mg: (15~35) mg.

5. The composition according to claim 1, characterized in that, The stabilizer includes at least one selected from tromethamine and tromethamine hydrochloride.

6. The composition according to claim 1, characterized in that, The molar ratio of the growth hormone fusion protein to the stabilizer is (20~90) mg: (0.01~0.05) mmol.

7. The composition according to claim 1, characterized in that, The molar ratio of the growth hormone fusion protein to the stabilizer is (50~70) mg: (0.015~0.035) mmol.

8. The composition according to claim 1, characterized in that, The molar ratio of the growth hormone fusion protein to the antioxidant is (20~90) mg: (0.01~0.02) mmol.

9. The composition according to claim 1, characterized in that, The molar ratio of the growth hormone fusion protein to the metal chelator is (20~90) mg: (0.00015~0.01) mmol.

10. The composition according to claim 1, characterized in that, The molar ratio of the growth hormone fusion protein to the metal chelator is (50~70) mg: (0.00015~0.0003) mmol.

11. The composition according to claim 1, characterized in that, The antioxidant is methionine.

12. The composition according to claim 1, characterized in that, The metal chelating agent is disodium edetate.

13. The composition according to claim 1, characterized in that, The mass ratio of the growth hormone fusion protein to the surfactant is (1~50):(0.1~1).

14. The composition according to claim 1, characterized in that, The surfactant includes at least one selected from Tween 80, Tween 20, and poloxamer P188.

15. The composition according to claim 1, characterized in that, The composition is an injection solution.

16. The composition according to claim 15, characterized in that, The pH value of the composition is 6.1-7.

2.

17. The composition according to claim 15, characterized in that, The pH value of the composition is 6.4 to 7.

2.

18. The composition according to claim 15, characterized in that, The concentration of the growth hormone fusion protein is 20-90 mg / mL.

19. The composition according to claim 15, characterized in that, The concentration of the growth hormone fusion protein is 50-70 mg / mL.

20. The composition according to claim 15, characterized in that, The concentration of the protective agent is 20~90 mg / mL.

21. The composition according to claim 15, characterized in that, The concentration of the protective agent is 30~70 mg / mL.

22. The composition according to claim 15, characterized in that, The concentration of the buffer is 5~40 mM.

23. The composition according to claim 15, characterized in that, The concentration of the buffer is 15~30 mM.

24. The composition according to claim 15, characterized in that, The concentration of the stabilizer is 10~50mM.

25. The composition according to claim 15, characterized in that, The concentration of the stabilizer is 15~35 mM.

26. The composition according to claim 1, characterized in that, The growth hormone fusion protein has an amino acid sequence as shown in any one of SEQ ID NO: 1 to 4.

27. An injection solution, characterized in that, include: Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, and protectant; Based on the total volume of the injection solution, the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, and the concentration of the protective agent is 40-90 mg / mL. The protective agent is sucrose, and the concentration of sucrose is 40-90 mg / mL; or, the protective agent is sucrose and trehalose, and the concentration of sucrose is 20-45 mg / mL and the concentration of trehalose is 20-45 mg / mL.

28. An injection solution, characterized in that, include: Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, methionine, and protectant; Based on the total volume of the injection solution, the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethorphan is 15-35 mM, the concentration of the protective agent is 40-90 mg / mL, and the concentration of methionine is 10-20 mM. The protective agent is sucrose, and the concentration of sucrose is 40-90 mg / mL; or, the protective agent is sucrose and trehalose, and the concentration of sucrose is 20-45 mg / mL and the concentration of trehalose is 20-45 mg / mL.

29. An injection solution, characterized in that, include: Growth hormone fusion protein, histidine, histidine hydrochloride, tromethamine, disodium edetate, and protectant; Wherein, based on the total volume of the injection solution, the concentration of the growth hormone fusion protein is 50-70 mg / mL, the total concentration of histidine and histidine hydrochloride is 15-30 mM, the concentration of tromethamine is 15-35 mM, the concentration of the protective agent is 40-90 mg / mL, and the concentration of disodium edetate is 0.15-1 mM; The protective agent is sucrose, and the concentration of sucrose is 40-90 mg / mL; or, the protective agent is sucrose and trehalose, and the concentration of sucrose is 20-45 mg / mL and the concentration of trehalose is 20-45 mg / mL.

30. Use of the composition according to any one of claims 1 to 26 or the injection according to any one of claims 27 to 29 in the preparation of a medicament for the treatment and / or prevention of growth hormone abnormality-related diseases; The growth hormone-related disorders are selected from at least one of the following: childhood growth hormone deficiency, idiopathic short stature, adult growth hormone deficiency, Turner syndrome, and Prad-Willi syndrome.

31. An injection device or container, characterized in that, include: The composition according to any one of claims 1 to 26 or the injection solution according to any one of claims 27 to 29.

32. The injection device or container according to claim 31, characterized in that, The injection device or container includes at least one selected from a container bottle and a syringe.

33. The injection device or container according to claim 32, characterized in that, The syringe includes at least one selected from auto-injectors and pre-filled syringes.

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