WO2023164898A1 - 一种重组融合蛋白的制剂 - Google Patents

一种重组融合蛋白的制剂 Download PDF

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WO2023164898A1
WO2023164898A1 PCT/CN2022/079096 CN2022079096W WO2023164898A1 WO 2023164898 A1 WO2023164898 A1 WO 2023164898A1 CN 2022079096 W CN2022079096 W CN 2022079096W WO 2023164898 A1 WO2023164898 A1 WO 2023164898A1
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Prior art keywords
fusion protein
recombinant fusion
buffer
composition
composition according
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PCT/CN2022/079096
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English (en)
French (fr)
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方国波
蔡则玲
吴珩
徐剑光
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上海赛金生物医药有限公司
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Priority to CN202280007365.XA priority Critical patent/CN117136196A/zh
Priority to PCT/CN2022/079096 priority patent/WO2023164898A1/zh
Publication of WO2023164898A1 publication Critical patent/WO2023164898A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration

Definitions

  • the invention relates to the field of pharmaceutical preparations, in particular to a recombinant fusion protein preparation.
  • RA Rheumatoid arthritis
  • TNFa pro-inflammatory cytokine
  • RANKL NF- ⁇ B ligand
  • OPG Osteoprotegerin osteoprotegrin
  • WO2021/143733A1 a recombinant fusion protein
  • chemical denaturation includes dissociation, oxidation, deamidation, isomerization, and polymerization of protein molecules, which are affected by the amino acids that make up the protein and the conditions (salt, pH, and temperature) of the solvent containing the protein.
  • Physical denaturation includes changes in tertiary structure, covalent/non-covalent aggregation and adhesion of monomers, and ionic force interactions between the charges attached to proteins and the charges attached to solutes or solvents. These chemical or physical denaturations will cause the protein to lose its original physicochemical properties and physiological activities.
  • Liquid preparations are also one of the storage methods for antibody drugs.
  • the buffer system in which they are located keeps the pH within a certain range.
  • Agents, surfactants, osmotic pressure regulators, and even antibacterial agents make the protein stable in this buffer system, but due to the different protein structures, each protein requires a different buffer system and stabilizer, unsuitable buffer system or stable Drugs can even reduce the stability of proteins, so there are differences in the composition of liquid preparations required for different protein drugs.
  • One object of the present invention is to provide a recombinant fusion protein composition with high stability.
  • Another object of the present invention is to provide a trial format for rapid formulation development of formulations.
  • a recombinant fusion protein composition comprising a recombinant fusion protein, a buffer and a stabilizer,
  • the recombinant fusion protein includes the following elements fused together: (a) TNF receptor or its active fragment; (b) OPG or its active fragment; and optionally (c) Fc fragment.
  • the buffer is selected from phosphate buffer, citrate buffer, or a combination thereof.
  • the stabilizer is selected from amino acids, sugar alcohols, inorganic salts, or combinations thereof.
  • the amino acid is selected from the group consisting of arginine (Arginine), glycine (Glycine), histidine (Histidine), or a combination thereof.
  • the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, maltose, sorbitol, or combinations thereof.
  • the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or combinations thereof.
  • the composition is a pharmaceutical composition.
  • the TNF receptor is selected from the group consisting of TNFR2, TNFR1, or a combination thereof.
  • the recombinant fusion protein has the structure of OPG-TNFR2-Fc from the N-terminus to the C-terminus.
  • amino acid sequence of the TNFR2 receptor is shown in SEQ ID NO.:1.
  • OPG amino acid sequence is shown in SEQ ID NO.:2.
  • amino acid sequence of the Fc fragment is shown in SEQ ID NO.:3.
  • amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.:4.
  • the concentration of the recombinant fusion protein is 2.5-50mg/mL, more preferably 5-50mg/mL, more preferably 10-40mg/mL, more preferably 15-35mg/mL, more preferably Preferably 20-25mg/mL, optimally 25mg/mL.
  • composition can also include surfactants, and the surfactants include (but not limited to): cationic surfactants, anionic surfactants, nonionic surfactants agent, or a combination thereof.
  • the surfactant is selected from the group consisting of polysorbate, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, poloxamer, or its combination.
  • the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40), polysorbate Ester 60 (PS-60, Tween-60), polysorbate 80 (PS-80, Tween-80), or a combination thereof.
  • the surfactant includes polysorbate 20 (PS-20, Tween-20).
  • the content of the surfactant is 0.001-8wt%, preferably 0.001-3wt%, more preferably 0.001-1wt%, more preferably 0.005-0.5wt%, more preferably 0.005- The composition of total weight.
  • the buffer is selected from the group consisting of citric acid-phosphate buffer, citric acid-sodium citrate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or combinations thereof.
  • the medium of the buffer solution is water.
  • the concentration of the buffer is 1-100mM, preferably 1-80mM, more preferably 1-50mM, more preferably 5-40mM, more preferably 5-25mM, more preferably 5-20 mM, optimally 10 mM.
  • the buffer includes citric acid-disodium hydrogen phosphate buffer.
  • the concentration of the citric acid-disodium hydrogen phosphate buffer solution is 1-100mM, preferably 1-80mM, more preferably 1-50mM, more preferably 5-40mM, more preferably 5 -25 mM, more preferably 5-20 mM, most preferably 10 mM.
  • the stabilizer has the effect of regulating osmotic pressure.
  • the stabilizer is selected from the group consisting of amino acids, sugar alcohols, inorganic salts, or combinations thereof.
  • the stabilizer includes sucrose.
  • the stabilizer includes sucrose, sodium chloride, arginine or amino acids with the same properties, or a combination thereof.
  • the content of the stabilizer is 0.5-50wt%, preferably 0.5-40wt%, more preferably 0.8-30wt%, more preferably 1-20wt%, more preferably 1-15wt% %, more preferably 1-10 wt%, more preferably 1-8 wt%, more preferably 2-7 wt%, most preferably 4 wt%, based on the total weight of the composition.
  • the concentration of the amino acid is 1-150mM, preferably 1-100mM, more preferably 10-80mM, more preferably 20-70mM, more preferably 40mM -60 mM, optimally 50 mM.
  • the content of said sugar alcohol is 0.5-15wt%, preferably 0.5-10wt%, more preferably 1-8wt%, more preferably 1.5-6wt%, more preferably Preferably 2-5 wt%, most preferably 3 wt%, based on the total weight of the composition.
  • the concentration of the inorganic salt is 10-200mM, preferably 30-150mM, more preferably 60-140mM, more preferably 70-130mM, more preferably 90-110 mM, optimally 100 mM.
  • the pH of the composition is 5.0-7.0, preferably 5.0-6.5, more preferably 5.2-6.4, more preferably 5.3-6.3, most preferably 5.4-6.0, such as 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • composition includes:
  • composition includes:
  • composition includes:
  • composition includes:
  • compositions as described in the first aspect of the present invention or its preparation for the preparation of medicaments for (i) preventing and/or treating infectious diseases and/or (ii) prevention and/or treatment of autoimmune diseases; and/or (iii) prevention and/or treatment of osteoporosis or loss; and/or (iv) prevention and/or treatment of tumor-related diseases.
  • the infectious disease includes septic shock (such as LPS-induced septic shock).
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, or a combination thereof.
  • osteoporosis or loss is selected from the group consisting of osteoporosis and/or loss caused by rheumatoid arthritis, osteoporosis in postmenopausal women, or a combination thereof.
  • the tumor-related disease is selected from the group consisting of multiple myeloma, bone-related solid tumor with bone metastases, or a combination thereof.
  • a method for preventing and/or treating a disease comprising the step of: administering the composition or its preparation as described in the first aspect of the present invention to a subject in need, thereby treating the disease, the disease selected from the group consisting of infectious disease, autoimmune disease, osteoporosis or loss, tumor-related disease, or a combination thereof.
  • Figure 1 shows the isometric diagram of the effect of aggregates, sodium chloride and sucrose after incubation at 50°C for 2 days.
  • Figure 2 shows the isometric diagram of the effect of polymers with polysorbate 20 and arginine after incubation at 50°C for 2 days.
  • Fig. 3 shows the contour diagram of the degradation and the effect of NaCl and sucrose after incubation at 50°C for 2 days.
  • Fig. 4 shows the contour map of the degradation and the effect of polysorbate 20 and arginine after incubation at 50°C for 2 days.
  • Figure 5 shows the isometric diagram of the effect of monomers with sodium chloride and sucrose after incubation at 50°C for 2 days.
  • Figure 6 shows the contour map of the effect of monomers, polysorbate 20 and arginine after incubation at 50°C for 2 days.
  • the inventors have developed a composition that can effectively maintain the stability of the recombinant fusion protein, and the recombinant fusion protein in the composition can maintain stability under high temperature conditions. Therefore, the composition of the present invention can provide a stable quantity of recombinant fusion protein, prolong the shelf life of the product, and improve the safety of clinical practice. On this basis, the present invention has been accomplished.
  • the terms “comprising”, “including”, and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • the weight content (wt.%) of each component is based on the weight of the composition.
  • compositions of the present invention include purified recombinant fusion proteins.
  • the recombinant fusion protein comprises the following elements fused together: (a) TNF receptor or active fragment thereof; (b) OPG or active fragment thereof; and optionally (c) Fc fragment.
  • the TNF receptor is selected from the group consisting of TNFR2, TNFR1, or a combination thereof.
  • the recombinant fusion protein of the present invention has the structure of OPG-TNFR2-Fc.
  • the TNFR2 receptor amino acid sequence of the present invention is as follows:
  • the OPG amino acid sequence of the present invention is as follows:
  • amino acid sequence of the Fc fragment of the present invention is as follows:
  • the recombinant fusion protein of the present invention contains (from N-terminal to C-terminal): OPG amino acid sequence 22-194, including 4 CRDs; TNFR2 amino acid sequence 23-257, including 4 CRDs; and IgG1Fc.
  • OPG amino acid sequence 22-194, including 4 CRDs TNFR2 amino acid sequence 23-257, including 4 CRDs
  • IgG1Fc IgG1Fc.
  • the term "recombinant fusion protein” also includes variant forms of the recombinant fusion protein (sequence shown in SEQ ID NO.:4) having the above activities. These variant forms include (but are not limited to): 1-3 (usually 1-2, more preferably 1) amino acid deletions, insertions and/or substitutions, and additions or substitutions at the C-terminal and/or N-terminal Deletion of one or several (usually within 3, preferably within 2, more preferably within 1) amino acids. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding or deleting one or several amino acids at the C-terminus and/or N-terminus usually does not change the structure and function of the protein.
  • the term also includes monomeric and multimeric forms of the polypeptides of the invention.
  • the term also includes linear as well as non-linear polypeptides (eg, cyclic peptides).
  • Recombinant fusion protein also includes active fragments, derivatives and analogs of the above-mentioned recombinant fusion protein.
  • fragment refers to a polypeptide that substantially retains the function or activity of the recombinant fusion protein of the present invention.
  • polypeptide fragments, derivatives or analogs of the present invention can be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more A polypeptide with substituent groups in amino acid residues, or (iii) a polypeptide formed by fusing an antigenic peptide to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence A polypeptide fused to this polypeptide sequence (recombinant fusion protein formed by fusing with a leader sequence, a secretory sequence or a tag sequence such as 6 ⁇ His).
  • conservative or non-conservative amino acid residues preferably conservative amino acid residues
  • amino acid residues preferably conservative amino acid residues
  • a polypeptide formed by fusing an antigenic peptide to another compound such as a compound that extends the half-life of the polypeptide
  • a preferred class of active derivatives refers to that compared with SEQ ID NO.: 4, at most 3, preferably at most 2, more preferably at most 1 amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.
  • the present invention also provides compositions containing analogs of the above-mentioned recombinant fusion proteins.
  • the difference between these analogs and the polypeptide shown in SEQ ID NO.: 4 may be a difference in the amino acid sequence, or a modification that does not affect the sequence, or both.
  • Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, ⁇ , ⁇ -amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.
  • Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during synthesis and processing or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.
  • chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications of polypeptides during
  • compositions and methods for their preparation are provided.
  • the invention provides a recombinant fusion protein composition, which comprises a recombinant fusion protein, a buffer and a stabilizer.
  • composition of the present invention is preferably a pharmaceutical composition.
  • composition of the present invention is preferably a liquid preparation, such as an oral liquid preparation or an injection liquid preparation.
  • the content of the recombinant fusion protein is 5-50mg/mL, more preferably 10-40mg/mL, more preferably 15-35mg/mL, more preferably 20-25mg/mL, Optimally 25 mg/mL.
  • the composition can also include surfactants, which include (but not limited to): positive ionic surfactants, anionic surfactants, nonionic A surfactant, or a combination thereof.
  • surfactants include (but not limited to): positive ionic surfactants, anionic surfactants, nonionic A surfactant, or a combination thereof.
  • the surfactant includes (but not limited to): polysorbate, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerin fatty acid ester, poloxamer, or combination.
  • the polysorbate includes (but not limited to): polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40), polysorbate 60 (PS-60, Tween-60), polysorbate 80 (PS-80, Tween-80), or a combination thereof.
  • the surfactant includes polysorbate 20 (PS-20, Tween-20).
  • the content of the surfactant is 0.001-8wt%, preferably 0.001-3wt%, more preferably 0.001-1wt%, more preferably 0.005-0.5wt%, more preferably 0.005-0.2wt%, more preferably 0.008-0.15wt%, more preferably 0.01-0.1wt%, more preferably 0.02-0.08wt%, more preferably 0.03-0.06wt%, most preferably 0.05wt%, with by total weight of the composition.
  • the buffer includes (but not limited to): phosphate buffer, citrate buffer, or a combination thereof.
  • the buffer includes (but not limited to): citric acid-disodium hydrogen phosphate buffer, citric acid-sodium citrate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer or its combination.
  • the medium of the buffer solution is water.
  • the concentration of the buffer is 1-100mM, preferably 1-80mM, more preferably 1-50mM, more preferably 5-40mM, more preferably 5-25mM, more preferably Preferably 5-20 mM, optimally 10 mM.
  • the buffer includes citric acid-disodium hydrogen phosphate buffer.
  • the concentration of the citrate buffer is 1-100mM, preferably 1-80mM, more preferably 1-50mM, more preferably 5-40mM, more preferably 5-25mM, More preferably 5-20 mM, most preferably 10 mM.
  • the stabilizers include (but not limited to) osmotic pressure regulators.
  • the stabilizer includes (but not limited to): amino acids, sugar alcohols, inorganic salts, or combinations thereof.
  • the amino acid includes (but not limited to): arginine (Arginine), glycine (Glycine), histidine (Histidine), or a combination thereof.
  • the sugar alcohols include (but not limited to): sucrose, mannitol, trehalose, maltose, sorbitol, or combinations thereof.
  • the inorganic salts include (but not limited to): sodium chloride, potassium chloride, calcium chloride, magnesium chloride, or combinations thereof.
  • the stabilizer includes sucrose.
  • the stabilizer includes: sucrose, sodium chloride, arginine or amino acids with the same properties, or a combination thereof.
  • the content of the stabilizer is 0.5-50wt%, preferably 0.5-40wt%, more preferably 0.8-30wt%, more preferably 1-20wt%, more preferably 1 - 15 wt%, more preferably 1-10 wt%, more preferably 1-8 wt%, more preferably 2-7 wt%, most preferably 4 wt%, based on the total weight of the composition.
  • the pH of the composition is 5.0-7.0, preferably 5.0-6.5, more preferably 5.2-6.4, more preferably 5.3-6.3, most preferably 5.4-6.0, for example 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • the composition includes:
  • recombinant fusion protein 10-40mg/mL Polysorbate 20 0-0.1wt% Citric acid-disodium hydrogen phosphate buffer 1-50mM Sodium chloride 0-140mM sucrose 0.5-15% by weight; and pH 5.0-7.0
  • the composition includes:
  • Citric acid-disodium hydrogen phosphate buffer 1-50mM Sodium chloride 10-140mM sucrose 0-10% by weight; and pH 5.0-7.0
  • the composition includes:
  • recombinant fusion protein 15-35mg/mL Polysorbate 20 0-0.08wt% Citric acid-disodium hydrogen phosphate buffer 5-25mM Sodium chloride 60-140mM sucrose 1.5-6% by weight; and pH 5.2-6.4
  • the composition includes:
  • recombinant fusion protein 20-25mg/mL Polysorbate 20 0.03-0.06wt% Citric acid-disodium hydrogen phosphate buffer 5-20mM Sodium chloride 90-110mM sucrose 2-5% by weight; and pH 5.2-6.4
  • the present invention also provides a method for preparing the composition of the present invention, the method comprising the steps of:
  • compositions provided by the invention are preferably in the form of liquid formulations.
  • the production of liquid preparations mainly replaces the protein into the designated buffer system by changing the liquid, which is more operable than the freeze-drying process, and the production cost is relatively lower than that of the freeze-drying process.
  • the present invention also provides a use of the composition according to the present invention or its preparation for the preparation of medicaments for (i) preventing and/or treating infectious diseases; and/or (ii) preventing and/or or treatment of autoimmune diseases; and/or (iii) prevention and/or treatment of osteoporosis or loss; and/or (iv) prevention and/or treatment of tumor-related diseases.
  • the infectious disease includes septic shock (such as LPS-induced septic shock).
  • the autoimmune disease is selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, or a combination thereof.
  • osteoporosis or loss is selected from the group consisting of osteoporosis and/or loss caused by rheumatoid arthritis, osteoporosis in postmenopausal women, or a combination thereof.
  • the tumor-related disease is selected from the group consisting of multiple myeloma, bone-related solid tumor with bone metastases, or a combination thereof.
  • the present invention also provides a method for preventing and/or treating a disease, comprising the step of: administering a composition as described in the present invention or its formulation to a subject in need, thereby treating the disease, the disease being selected from the group consisting of infection disease, autoimmune disease, osteoporosis or loss, tumor-related disease, or a combination thereof.
  • step (ii) the composition is kept at 50° C. for ⁇ 3 days, preferably 2 days, more preferably 1 day.
  • the stability of the detection composition includes the aggregate ratio, monomer ratio and degradation ratio of the detection composition.
  • polymer ratio ⁇ 15% preferably ⁇ 10%, more preferably ⁇ 7.5%), degradation ratio ⁇ 15% (preferably ⁇ 10%, more preferably ⁇ 7.5%), and If the ratio of monomers is ⁇ 70% (preferably ⁇ 80%, more preferably ⁇ 85%), it is determined that the component to be screened and its concentration are suitable for the composition described in the first aspect of the present invention.
  • the present invention adopts the high temperature exposure test to carry out the stability accelerated test on the sample, which is different from the commonly used 40°C. According to the nature of the protein, the present invention increases the accelerated temperature to 50°C, which further accelerates the change speed of protein stability and greatly shortens the protein preparation. Developmental test cycle.
  • the present invention adopts the combination of orthogonal test and DOE (experimental design) to study the stability of the protein, and quickly and effectively screen the prescription of the preparation.
  • composition containing the recombinant fusion protein provided by the present invention can improve the stability of the recombinant fusion protein.
  • the present invention provides an inspection method capable of more efficiently detecting the thermal stability of a composition.
  • citrate buffer and phosphate buffer in the following examples are as follows:
  • Citric acid buffer the medium is water, the solute is citric acid and disodium hydrogen phosphate.
  • Phosphate buffer the medium is water, and the solutes are sodium dihydrogen phosphate and disodium hydrogen phosphate.
  • the recombinant fusion protein sequence in the following examples is shown in SEQ ID NO.:4.
  • SEC-HPLC is a size exclusion chromatography, which is used to analyze the purity of the recombinant fusion protein, specifically, to detect the content of polymers produced by aggregation and the content of small molecular fragments produced by degradation of the recombinant fusion protein.
  • the above-mentioned different pH refers to 10 mM citric acid-disodium hydrogen phosphate buffer solution of pH 3.0, pH 4.0, pH 5.0, pH 6.0, pH 7.0, and pH 8.0.
  • sample preparations were respectively exchanged into 10 mM citric acid-disodium hydrogen phosphate buffer solution with different pH, exposed at 50° C. for 3 days, and then analyzed by SE-HPLC.
  • Table 2 shows the test results of zero-point samples for pH stability screening. The results of pH stability screening at 50°C for 3 days for sample purity are shown in Table 3.
  • the stability of the sample is relatively best at pH 6.0. This example basically determines the stable pH range of the target protein, and the preferred stable pH range should be higher than 5.0, about 5.5 to 6.5.
  • Example 1 According to the pH range determined in Example 1, the stability of the recombinant fusion protein in phosphate buffer and citrate buffer system was investigated, and a suitable buffer solution was screened.
  • the pH range of phosphate buffer and citric acid buffer is 5.5-6.5
  • the concentration of the buffer system is 10-50mM
  • the protein concentration is 25mg/ml.
  • DOE design is adopted, according to the pH and the concentration of the buffer solution. Factors to design formulation prescriptions. Preparation samples were incubated under accelerated stability test conditions (50°C), samples were taken after 1 day and 2 days respectively, and SEC detection was performed to compare and investigate the stability of samples under different conditions.
  • Results See Table 4 for the prescription test results of the phosphate buffer system preparation, and Table 5 for the prescription test results of the citric acid buffer system preparation.
  • the stability of the sample was poor, and it degraded violently under the accelerated condition of high temperature, and the degradation rate was high under different concentrations and different pH conditions. This result indicates that the sample is not suitable for long-term storage in the phosphate buffer system.
  • the stability of the sample under high-temperature accelerated conditions is relatively good, and its polymer content is slightly higher than that of the phosphate system sample, but the degradation content is much lower than that of the phosphate system sample.
  • the sample The stability in citrate buffer is higher than that in phosphate buffer.
  • the use of phosphate buffer as the buffer system of the sample is basically excluded, and the subsequent stability research is carried out on the premise of using the citric acid buffer system as the basic buffer system.
  • citric acid buffer solution further investigate the concentration and pH of the buffer solution, and design the DOE prescription with 2 factors and 3 levels. 25mg/mL. Preparation samples were investigated under accelerated test conditions, and after incubation at 50°C for 1 day, the protein purity was determined.
  • the measurement results are shown in Table 6.
  • the results show that the aggregation degree of the sample is affected by the concentration of citrate buffer and pH. In the range of pH5.5-pH6.5, the aggregate content increases with the increase of pH, and decreases with the increase of the concentration of citrate buffer. , indicating that the relatively low pH in this range and the relatively high concentration of citrate buffer can inhibit the aggregation of recombinant fusion proteins.
  • the degradation of the sample in this example has a tendency to decrease with the increase of pH, but the change is relatively small; the change of the concentration of citric acid buffer has no obvious influence on the degradation content of the sample. Therefore, within the pH and concentration range of the present embodiment, the citric acid buffer concentration and pH have less impact on sample degradation
  • the monomer content of the sample in this example decreases with the increase of pH, and when the pH is less than or equal to 6.0, the content is relatively stable and the change is small, but when the pH is greater than 6.0, the monomer content decreases significantly, indicating that When the pH is greater than 6.0, the stability of the sample is relatively poor, and the monomer content tends to increase with the increase of the citrate buffer concentration.
  • citrate buffer was determined as the basic buffer of the sample. Further, through the buffer concentration and pH range confirmation test, the influence of the citric acid buffer concentration and pH on the stability of the sample was investigated, and the analysis was carried out from the three aspects of polymer, monomer and degradation, and it was confirmed that the sample was at pH5.5-pH6. The internal stability of citrate buffer of 5 is good.
  • a more preferred pH range is pH5.5-pH6.0, and a more preferred buffer concentration is a high-concentration citric acid buffer, with 50 mM citric acid being the most optimal.
  • the final product of the sample is used as a drug for subcutaneous injection, and the content of citric acid needs to be reduced to reduce injection irritation.
  • the stability effect of high-concentration citric acid buffer on the sample is better than that of low-concentration citric acid buffer. It is considered that the concentration of salt ions in high-concentration citric acid buffer is higher, thereby increasing the stability of the sample, while the concentration of salt ions It can be improved by adding other excipients in subsequent preparations. Therefore, the concentration of citric acid in the sample buffer can be reduced to 10 mM to reduce injection irritation.
  • the optimal buffer system for the sample is 10 mM citrate buffer, pH 5.5 to pH 6.0.
  • Example 2 the 10mM pH5.5-6.0 citrate buffer solution determined in Example 2 was used to investigate the effect of the added excipients on protein stability when the buffer solution was determined. Choose one representative excipient from each of the four types of excipients commonly used to stabilize proteins: sugar alcohols, salts, surfactants, and amino acids, and conduct DOE experimental design to quickly investigate the stability of samples through high-temperature accelerated tests. stability.
  • Sucrose was selected as the excipient for sugar alcohols, NaCl was selected for salts, polysorbate 20 was selected for surfactants, and arginine was selected for amino acids.
  • concentration range of sucrose is 0-10%, the concentration range of NaCl is 0-140mM, the concentration range of arginine is 0-100mM, and the DOE design is carried out at two levels of 4 factors. Center repeats.
  • Test results are shown in Table 8 and Table 9.
  • the DOE statistical analysis was performed on the purity test data after incubation at 50°C for 2 days in Table 9, the model was statistically significant, p ⁇ 0.05.
  • Figure 2 The equivalent diagrams of the effects of the above four excipients on degradation after 2 days of incubation at 50°C are shown in Figures 3 and 4.
  • Figures 5 and 6 The isopleth diagrams of the effects of the above four excipients on monomers after 2 days of incubation at 50°C are shown in Figures 5 and 6.
  • the concentration change of polysorbate 20 did not affect the aggregation, but the addition of arginine had a certain aggregation inhibitory effect, the higher the arginine content, the lower the aggregation content. Comparing the experimental groups 1 and 2 and the experimental groups 3 and 4 in Table 8, it can be seen that in the preparation not containing NaCl, arginine plays an obvious aggregation inhibitory effect, but after adding 140mM NaCl in the preparation, adding arginine has no effect. The effect of inhibiting aggregation shows that the addition of arginine only plays the role of increasing the concentration of salt ions to increase the stability of the sample, and itself has no obvious aggregation inhibiting effect on the sample.
  • the test results in the above table and Figure 6 show that the addition of arginine has a tendency to increase the monomer content, which is related to the increase of the salt ion concentration and the inhibition of the polymer.
  • the addition of polysorbate 20 can slightly increase the monomer content, but increases The amount is less, showing that polysorbate 20 has little influence on purity. From the perspective of cost and process complexity, polysorbate 20 and similar surfactants may not be added in the preparation.
  • the optimal condition is to add 0.05% polysorbate Ester 20, can further improve the stability of the sample.
  • DOE data analysis was carried out from the perspectives of polymers, degradation and monomers, and it was found that sugar alcohols and salt ion excipients mainly play a role in protein stability, here mainly refer to sucrose and chlorinated Sodium, but not limited to these two, addition of surfactants can slightly inhibit the degradation of the sample, including but not limited to polysorbate 20.
  • Embodiment 4 preparation prescription screening
  • the preferred formulation of this sample is a different combination of citrate buffer, sugar alcohols, salt ions, and surfactant-based excipients, with a pH of 5.2 to 6.4.
  • concentration of the citrate buffer is 10 to 50 mM
  • the sugar alcohols include but not limited to sucrose
  • the salt ions include but not limited to sodium chloride and some amino acids
  • surfactants include but not limited to polysorbate 20.

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Abstract

本发明涉及一种重组融合蛋白制剂。具体地，本发明提供一种重组融合蛋白组合物，所述的组合物包括OPG-TNFR2-Fc重组融合蛋白、缓冲液和稳定剂。本发明所述的组合物能够提高OPG-TNFR2-Fc重组融合蛋白的稳定性。

Description

一种重组融合蛋白的制剂 技术领域
本发明涉及药物制剂领域，具体地涉及一种重组融合蛋白制剂。
背景技术
自身免疫性疾病是一种免疫介导的疾病，是免疫系统对宿主自身抗原发生正性应答、造成组织或器官的病理性损伤、影响其生理功能、并最终导致各种临床症状的状态。类风湿性关节炎(RA)是自身免疫性疾病中最常见、发病率最高的一种，它对滑膜、关节软骨和骨骼造成伤害。RA是由一系列促炎细胞因子通过相互作用而引起并维持的，其中，TNFα是炎症性关节炎患者关键的炎症介导细胞因子。目前有多种抑制TNFa功能的药物(TNFa阻断剂)在临床上治疗各种自身免疫性疾病。
关节炎是慢性软组织炎症，最后通常伴随着关节的破坏。免疫介导的关节炎通常会造成炎症关节内的局灶性骨糜烂，这主要是由于受体激活剂NF-κB配体(RANKL)介导的破骨细胞活性过多而引起。RANKL是骨吸收的重要介质，骨保护素osteoprotegrin(OPG)是RANKL的诱饵受体、能特异性结合RANKL的可溶性蛋白，是RANKL的生理抑制剂。
然而，目前的药物虽然能改善RA患者的炎症，但对有炎症关节的局灶性骨侵蚀和全身性骨丢失没有或很有限的预防和治疗作用。
本发明人的前期研究提供了一种重组融合蛋白(WO2021/143733A1)，其既能改善RA患者的炎症，也能防止炎症造成的局灶性骨糜烂和全身性骨丢失的药物。
通常，由于不利的温度、剪切力、振动、冻融、UV暴露、过度pH变化、有机溶剂、微生物污染等因素容易导致蛋白质药物的化学和物理性质的变化。其中化学变性包括蛋白分子的解离、氧化、脱酰胺、异构化和聚合，其受到组成蛋白质的氨基酸以及含有该蛋白质的溶剂的条件(盐、pH和温度)的影响。物理变性包括三级结构的变化、单体的共价/非共价聚集和粘附，蛋白上附带的电荷与溶质或溶剂自带的电荷之间发生的离子力作用。这些化学或物理变性均会使得蛋白失去其原有的理化性质和生理活性。
作为生物药物，蛋白变性会对药物的药效、安全性等产生巨大影响，例如会导致病人的免疫应答反应，因此一种能够维持蛋白理化性质，使得蛋白能够长期稳定储存的液体制剂对蛋白药物的质量极其重要。
市面上已有多个生物抗体药物采用冻干的方式进行药物的长期储存，液体制剂也是抗体药物储存的方式之一，其所在的缓冲体系使得pH维持在一定范围内，向其中添加各种稳定剂、表面活性剂、渗透压调节剂甚至抗菌剂等使得蛋白在此缓冲体系中保持稳定，但由于蛋白结构的不同，每种蛋白需要不同的缓冲体系及稳定剂，不适宜的缓冲体系或稳定剂甚至会降低蛋白的稳定性，因此不同的蛋白药物所需要的液体制剂组成成分之前存在差异。
因此，本领域需要开发一种具有高稳定性的重组融合蛋白制剂，提高重组融合蛋白的稳定性。
发明内容
本发明的一个目的是提供一种具有高稳定性的重组融合蛋白组合物。
本发明的另一个目标是提供快速制剂处方开发的试验方式。
在本发明的第一方面，提供了一种重组融合蛋白组合物，所述的组合物包括重组融合蛋白、缓冲液和稳定剂，
其中，所述重组融合蛋白包括融合在一起的以下元件：(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc片段。
在另一优选例中，所述的缓冲液选自磷酸盐缓冲液、柠檬酸缓冲液、或其组合。
在另一优选例中，所述的稳定剂选自氨基酸、糖醇、无机盐，或其组合。
在另一优选例中，所述的氨基酸选自下组：精氨酸(Arginine)、甘氨酸(Glycine)、组氨酸(Histidine)，或其组合。
在另一优选例中，所述的糖醇选自下组：蔗糖(Sucrose)、甘露醇(Mannitol)、海藻糖(Trehalose)、麦芽糖(Maltose)、山梨醇(Sorbitol)，或其组合。
在另一优选例中，所述的无机盐选自下组：氯化钠、氯化钾、氯化钙、氯化镁，或其组合。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的TNF受体选自下组：TNFR2、TNFR1、或其组合。
在另一优选例中，所述的重组融合蛋白从N端到C端具有OPG-TNFR2-Fc的结构。
在另一优选例中，所述的TNFR2受体氨基酸序列如SEQ ID NO.:1所示。
在另一优选例中，所述的OPG氨基酸序列如SEQ ID NO.:2所示。
在另一优选例中，所述的Fc片段氨基酸序列如SEQ ID NO.:3所示。
在另一优选例中，所述的重组融合蛋白氨基酸序列如SEQ ID NO.:4所示。
在另一优选例中，所述重组融合蛋白的浓度为2.5-50mg/mL，更佳地5-50mg/mL，更佳地10-40mg/mL，更佳地15-35mg/mL，更佳地20-25mg/mL，最佳地25mg/mL。
在另一优选例中，所述的组合物还可以包括表面活性剂，所述的表面活性剂包括(但不限于)：正离子型表面活性剂、负离子型表面活性剂、非离子型表面活性剂，或其组合。
在另一优选例中，所述的表面活性剂选自下组：聚山梨酯、聚氧乙烯山梨醇酐脂肪酸酯、聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、泊洛沙姆，或其组合。
在另一优选例中，所述的聚山梨酯选自下组：聚山梨酯20(PS–20，吐温-20)、聚山梨酯40(PS-40，吐温-40)、聚山梨酯60(PS–60，吐温-60)、聚山梨酯80(PS-80，吐温-80)，或其组合。
在另一优选例中，所述的表面活性剂包括聚山梨酯20(PS-20，吐温-20)。
在另一优选例中，所述表面活性剂的含量为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.005-0.5wt％，更佳地0.005-0.2wt％，更佳地0.008-0.15wt％，更佳地0.01-0.1wt％，更佳地0.02-0.08wt％，更佳地0.03-0.06wt％，最佳地0.05wt％，以组合物的总重量计。
在另一优选例中，所述的缓冲液选自下组：柠檬酸-磷酸缓冲液、柠檬酸-柠檬酸钠缓冲液、磷酸二氢钠-磷酸氢二钠缓冲液或其组合。
在另一优选例中，所述的缓冲液的介质为水。
在另一优选例中，所述缓冲液的浓度为1-100mM，较佳地为1-80mM，更佳地1-50mM，更佳地5-40mM，更佳地5-25mM，更佳地5-20mM，最佳地10mM。
在另一优选例中，所述的缓冲液包括柠檬酸-磷酸氢二钠缓冲液。
在另一优选例中，所述柠檬酸-磷酸氢二钠缓冲液的浓度为1-100mM，较佳地1-80mM，更佳地1-50mM，更佳地5-40mM，更佳地5-25mM，更佳地5-20mM，最佳地10mM。
在另一优选例中，所述的稳定剂具有渗透压调节作用。
在另一优选例中，所述的稳定剂选自下组：氨基酸类、糖醇类、无机盐类，或其组合。
在另一优选例中，所述的稳定剂包括蔗糖。
在另一优选例中，所述的稳定剂包括蔗糖、氯化钠、精氨酸或与其性质相同的氨 基酸、或其组合。
在另一优选例中，所述的稳定剂的含量为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％，以组合物的总重量计。
在另一优选例中，所述的氨基酸(优选为精氨酸)的浓度为1-150mM，较佳地1-100mM，更佳地10-80mM，更佳地20-70mM，更佳地40-60mM，最佳地50mM。
在另一优选例中，所述的糖醇(优选为蔗糖)的含量为0.5-15wt％，较佳地0.5-10wt％，更佳地1-8wt％，更佳地1.5-6wt％，更佳地2-5wt％，最佳地3wt％，以组合物的总重量计。
在另一优选例中，所述的无机盐(优选为氯化钠)的浓度为10-200mM，较佳地30-150mM，更佳地60-140mM，更佳地70-130mM，更佳地90-110mM，最佳地100mM。
在另一优选例中，所述的组合物的pH为5.0-7.0，较佳地5.0-6.5，更佳地5.2-6.4，更佳地5.3-6.3，最佳地5.4-6.0，例如5.4、5.5、5.6、5.7、5.8、5.9、或6.0。
在另一优选例中，所述的组合物包括：
Figure PCTCN2022079096-appb-000001
在另一优选例中，所述的组合物包括：
Figure PCTCN2022079096-appb-000002
在另一优选例中，所述的组合物包括：
Figure PCTCN2022079096-appb-000003
Figure PCTCN2022079096-appb-000004
在另一优选例中，所述的组合物包括：
Figure PCTCN2022079096-appb-000005
在本发明的第二方面，提供了一种如本发明第一方面所述的组合物或其制剂的用途，用于制备药物，所述药物用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或治疗肿瘤相关疾病。
在另一优选例中，所述感染性疾病包括感染性休克(比如LPS诱导的感染性休克)。
在另一优选例中，所述自身免疫性疾病选自下组：类风湿性关节炎、强直性脊柱炎、或其组合。
在另一优选例中，骨质疏松或丢失选自下组：类风湿性关节炎引起的骨质疏松或/和丢失、妇女绝经后的骨质疏松、或其组合。
在另一优选例中，所述肿瘤相关疾病选自下组：多发性骨髓瘤、骨相关的骨转移实体瘤、或其组合。
在本发明的第三方面，提供了一种预防和/或治疗疾病的方法，包括步骤：给需要的对象施用如本发明第一方面所述组合物或其制剂，从而治疗疾病，所述疾病选自下组：感染性疾病、自身免疫性疾病、骨质疏松或丢失、肿瘤相关疾病、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具 体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了50℃孵育2天后聚体与氯化钠及蔗糖影响等值图。
图2显示了50℃孵育2天后聚体与聚山梨酯20及精氨酸影响等值图。
图3显示了50℃孵育2天后降解与NaCl及蔗糖影响等值图。
图4显示了50℃孵育2天后降解与聚山梨酯20及精氨酸影响等值图。
图5显示了50℃孵育2天后单体与氯化钠及蔗糖影响等值图。
图6显示了50℃孵育2天后单体与聚山梨酯20及精氨酸影响等值图。
具体实施方式
本发明人通过广泛而深入的研究，研发出一种组合物，所述的组合物能够有效保持重组融合蛋白的稳定性，组合物中的重组融合蛋白能够在高温条件下保持稳定性。因此，本发明所述的组合物能够提供稳定的重组融合蛋白质量，延长产品的货架期，提高临床实用的安全性。在此基础上，完成了本发明。
术语
除非另有定义,否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。
如本文所用，“mM”为mmol/L单位，例如，1mM＝1mmol/L。
在本发明中在所述的组合物中，各个组分的重量含量(wt.％)均以组合物的重量计。
重组融合蛋白
在本发明中，本发明的组合物包括纯化的重组融合蛋白。所述重组融合蛋白包括融合在一起的以下元件：(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc片段。所述TNF受体选自下组：TNFR2、TNFR1、或其组合。优选地，本发 明的重组融合蛋白具有OPG-TNFR2-Fc的结构。
本发明所述的TNFR2受体氨基酸序列如下所示：
Figure PCTCN2022079096-appb-000006
本发明所述的OPG氨基酸序列如下所示：
Figure PCTCN2022079096-appb-000007
本发明所述的Fc片段氨基酸序列如下所示：
Figure PCTCN2022079096-appb-000008
优选地，本发明的重组融合蛋白含有(从N端到C端)：OPG氨基酸序列第22-194位，包括4个CRDs；TNFR2氨基酸序列第23-257位，包括4个CRDs；以及IgG1Fc。该重组融合蛋白(OPG-TNFR2-Fc融合蛋白)氨基酸序列如下所示：
Figure PCTCN2022079096-appb-000009
Figure PCTCN2022079096-appb-000010
如本文所用，术语“重组融合蛋白”还包括具有上述活性的重组融合蛋白(如SEQ ID NO.:4所示的序列)的变异形式。这些变异形式包括(但并不限于)：1-3个(通常为1-2个，更佳地1个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为3个以内，较佳地为2个以内，更佳地为1个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加或缺失一个或数个氨基酸通常也不会改变蛋白质的结构和功能。此外，所述术语还包括单体和多聚体形式的本发明多肽。该术语还包括线性以及非线性的多肽(如环肽)。
“重组融合蛋白”还包括上述重组融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明重组融合蛋白的功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)抗原肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6×His等标签序列融合而形成的重组融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与SEQ ID NO.:4相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供含有上述重组融合蛋白的类似物的组合物。这些类似物与SEQ ID NO.:4所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
组合物及其制备方法
本发明提供一种重组融合蛋白组合物，所述的组合物包括重组融合蛋白、缓冲液、稳定剂。
本发明所述的组合物优选为药物组合物。
本发明所述的组合物优选为液体制剂，例如口服液体制剂或注射液体制剂。
在本发明的一个优选例中，所述重组融合蛋白的含量为5-50mg/mL，更佳地10-40mg/mL，更佳地15-35mg/mL，更佳地20-25mg/mL，最佳地25mg/mL。
在本发明的一个优选例中，所述的组合物还可以包括表面活性剂，所述的表面活性剂包括(但不限于)：正离子型表面活性剂、负离子型表面活性剂、非离子型表面活性剂，或其组合。
代表性地，所述的表面活性剂包括(但不限于)：聚山梨酯、聚氧乙烯山梨醇酐脂肪酸酯、聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、泊洛沙姆，或其组合。
典型地，所述的聚山梨酯包括(但不限于)：聚山梨酯20(PS–20，吐温-20)、聚山梨酯40(PS-40，吐温-40)、聚山梨酯60(PS–60，吐温-60)、聚山梨酯80(PS-80，吐温-80)，或其组合。
典型地，所述的表面活性剂包括聚山梨酯20(PS-20，吐温-20)。
在本发明的一个优选例中，所述表面活性剂的含量为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.005-0.5wt％，更佳地0.005-0.2wt％，更佳地0.008-0.15wt％，更佳地0.01-0.1wt％，更佳地0.02-0.08wt％，更佳地0.03-0.06wt％，最佳地0.05wt％，以组合物的总重量计。
在本发明的一个优选例中，所述的缓冲液包括(但不限于)：磷酸盐缓冲液、柠檬酸缓冲液，或其组合。
在另一优选例中，所述的缓冲液包括(但不限于)：柠檬酸-磷酸氢二钠缓冲液、柠檬酸-柠檬酸钠缓冲液、磷酸二氢钠-磷酸氢二钠缓冲液或其组合。
在另一优选例中，所述的缓冲液的介质为水。
在本发明的一个优选例中，所述缓冲液的浓度为1-100mM，较佳地1-80mM，更佳地1-50mM，更佳地5-40mM，更佳地5-25mM，更佳地5-20mM，最佳地10mM。
代表性地，所述的缓冲液包括柠檬酸-磷酸氢二钠缓冲液。
在本发明的一个优选例中，所述柠檬酸缓冲液的浓度为1-100mM，较佳地1-80mM，更佳地1-50mM，更佳地5-40mM，更佳地5-25mM，更佳地5-20mM，最佳地10mM。
在本发明的一个优选例中，所述的稳定剂包括(但不限于)渗透压调节剂。
在本发明的一个优选例中，所述的稳定剂包括(但不限于)：氨基酸类、糖醇类、无机盐类，或其组合。
代表性地，所述的氨基酸包括(但不限于)：精氨酸(Arginine)、甘氨酸(Glycine)、组氨酸(Histidine)，或其组合。
代表性地，所述的糖醇包括(但不限于)：蔗糖(Sucrose)、甘露醇(Mannitol)、海藻糖(Trehalose)、麦芽糖(Maltose)、山梨醇(Sorbitol)，或其组合。
在本发明的一个优选例中，所述的无机盐包括(但不限于)：氯化钠、氯化钾、氯化钙、氯化镁，或其组合。
在另一优选例中，所述的稳定剂包括蔗糖。
代表性地，所述的稳定剂包括：蔗糖、氯化钠、精氨酸或与其性质相同的氨基酸、或其组合。
在本发明的一个优选例中，所述的稳定剂的含量为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％，以组合物的总重量计。
在本发明的一个优选例中，所述的组合物的pH为5.0-7.0，较佳地5.0-6.5，更佳地5.2-6.4，更佳地5.3-6.3，最佳地5.4-6.0，例如5.4、5.5、5.6、5.7、5.8、5.9、或6.0。
在本发明的一个优选例中，所述的组合物包括：
重组融合蛋白 10-40mg/mL
聚山梨酯20 0-0.1wt％
柠檬酸-磷酸氢二钠缓冲液 1-50mM
氯化钠 0-140mM
蔗糖 0.5-15wt％；和
pH 5.0-7.0
在本发明的一个优选例中，所述的组合物包括：
重组融合蛋白 10-40mg/mL
聚山梨酯20 0-0.1wt％
柠檬酸-磷酸氢二钠缓冲液 1-50mM
氯化钠 10-140mM
蔗糖 0-10wt％；和
pH 5.0-7.0
在本发明的一个优选例中，所述的组合物包括：
重组融合蛋白 15-35mg/mL
聚山梨酯20 0-0.08wt％
柠檬酸-磷酸氢二钠缓冲液 5-25mM
氯化钠 60-140mM
蔗糖 1.5-6wt％；和
pH 5.2-6.4
在本发明的一个优选例中，所述的组合物包括：
重组融合蛋白 20-25mg/mL
聚山梨酯20 0.03-0.06wt％
柠檬酸-磷酸氢二钠缓冲液 5-20mM
氯化钠 90-110mM
蔗糖 2-5wt％；和
pH 5.2-6.4
本发明还提供一种本发明所述组合物的制备方法，所述的方法包括步骤：
将如本发明所述的组合物的各组分混合后，得到重组融合蛋白组合物。
本发明提供的组合物优选为液体制剂形式。液体制剂的生产主要通过换液的方式将蛋白置换至指定的缓冲体系中，相对于冻干工艺，更具有操作性，且生产成本也相对低于冻干工艺生产成本。
用途
本发明还提供一种如本发明所述组合物或其制剂的用途，用于制备药物，所述药物用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或治疗肿瘤相关疾病。
在另一优选例中，所述感染性疾病包括感染性休克(比如LPS诱导的感染性休 克)。
在另一优选例中，所述自身免疫性疾病选自下组：类风湿性关节炎、强直性脊柱炎、或其组合。
在另一优选例中，骨质疏松或丢失选自下组：类风湿性关节炎引起的骨质疏松或/和丢失、妇女绝经后的骨质疏松、或其组合。
在另一优选例中，所述肿瘤相关疾病选自下组：多发性骨髓瘤、骨相关的骨转移实体瘤、或其组合。
本发明还提供了一种预防和/或治疗疾病的方法，包括步骤：给需要的对象施用施用如如本发明所述组合物或其制剂，从而治疗疾病，所述疾病选自下组：感染性疾病、自身免疫性疾病、骨质疏松或丢失、肿瘤相关疾病、或其组合。
检测方法
在本发明的另一方面，提供了一种用于筛选适用于本发明第一方面所述组合物的成分及其浓度的方法，所述方法包括步骤：
(i)提供所述的重组蛋白，并将一定浓度的待筛选成分与所述重组蛋白混合形成组合物；
(ii)将所述组合物保持在50℃下；和
(iii)检测所述组合物的稳定性，从而判定待筛选成分及浓度是否适用于本发明第一方面所述组合物。
在另一优选例中，在步骤(ii)中，将所述组合物保持在50℃下≤3天，较佳地2天，更佳地1天。
在另一优选例中，所述的检测组合物稳定性包括检测组合物的聚体比例、单体比例和降解比例。
在另一优选例中，如聚体比例≤15％(优选地≤10％，更优选地≤7.5％)，降解比例≤15％(优选地≤10％，更优选地≤7.5％)，并且单体比例≥70％(优选地≥80％，更优选地≥85％)，则判定该待筛选成分及浓度适用于本发明第一方面所述组合物。
本发明采用高温暴露试验对样品进行稳定性加速试验，不同于常用的40℃，本发明根据蛋白的性质，将加速温度提高至50℃，进一步加速了蛋白稳定性的变化速度，大大缩短蛋白制剂开发的试验周期。
本发明采用正交试验及DOE(试验设计)结合的方式，对蛋白的稳定性进行研究，快速有效地进行制剂处方筛选。
本发明的主要优点包括：
(1)本发明提供的含有重组融合蛋白的组合物，能够提高重组融合蛋白的稳定性。
(2)本发明提供了能够更高效检测组合物热稳定性的检验方法。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
以下实施例中的柠檬酸缓冲液和磷酸盐缓冲液如下：
柠檬酸缓冲液：介质为水，溶质为柠檬酸和磷酸氢二钠。
磷酸盐缓冲液：介质为水，溶质为磷酸二氢钠和磷酸氢二钠。
以下实施例中的重组融合蛋白序列如SEQ ID NO.:4所示。
通用测定方法
SEC-HPLC为体积排阻色谱法，用于分析重组融合蛋白的纯度，具体地，检测重组融合蛋白的因聚集产生的多聚体含量及降解产生的小分子片段含量。
实施例1 蛋白稳定性pH范围筛选
在本实施例中，研究样品在组成成分相同(均为柠檬酸-磷酸氢二钠)但pH不同的10mM柠檬酸-磷酸氢二钠缓冲体系中蛋白的稳定性。
上述不同pH是指pH3.0，pH4.0，pH5.0，pH6.0，pH7.0，pH8.0的10mM柠檬酸-磷酸氢二钠缓冲液。
蛋白换液至对应缓冲体系后分别在2-8℃及50℃下研究蛋白的稳定性。
样品制剂分别换液至不同pH的10mM柠檬酸-磷酸氢二钠缓冲液中，在50℃暴露3天，然后通过SE-HPLC进行分析。
结果：pH稳定性筛选零点样品检测结果如表2所示。pH稳定性筛选50℃3天样 品纯度检测结果如表3所示。
表2：pH稳定性筛选零点样品检测结果
Figure PCTCN2022079096-appb-000011
表3：pH稳定性筛选50℃3天样品纯度检测结果
Figure PCTCN2022079096-appb-000012
表2分析结果显示，室温条件下各制剂均相对稳定，无明显沉淀产生。表3的SE-HPLC分析结果显示，50℃加速时，样品pH为3、4时样品发生大量聚集。pH5.0时纯度相对较高，然而在检测时发现，pH5.0样品在检测过程中稀释处理时产生沉淀，而检测时样品过滤操作去除了沉淀。因此导致pH5样品检测纯度较好的实际原因是样品产生沉淀并经过滤去除。在pH5.0时样品实际稳定性较差，容易产生沉淀。该结果说明样品在低于或等于pH5环境下稳定性相对较差，趋向于聚集，易产生沉淀。
pH高于5.0时，样品高温加速条件下，样品的聚体含量随pH的上升而增加，而降解随pH的上升而减少，在pH6.0时样品的稳定性相对最好。本实施例基本确定了目标蛋白的稳定性pH范围，优选的稳定性pH范围应高于5.0，约为5.5到6.5。
实施例2 缓冲体系的筛选
2.1缓冲液筛选
根据实例1确定的pH范围，考察重组融合蛋白在磷酸盐缓冲液及柠檬酸缓冲液体系中的稳定性，筛选合适的缓冲溶液。
根据缓冲溶液特性，磷酸盐缓冲液和柠檬酸缓冲液pH范围均为5.5～6.5，缓冲体系浓度为10到50mM，蛋白浓度均为25mg/ml，采用DOE设计，按照pH、缓冲溶液浓度2个因素进行设计制剂处方。制剂样品在加速稳定性试验条件(50℃)孵育，分别于1天和2天后取样，进行SEC检测，对比考察不同条件下样品的稳定性。
结果：磷酸盐缓冲体系制剂处方试验结果见表4、柠檬酸缓冲体系制剂处方试验结果见表5。
表4 磷酸盐缓冲液体系的制剂蛋白纯度检测结果
Figure PCTCN2022079096-appb-000013
表5 柠檬酸缓冲液体系的制剂蛋白纯度检测结果
Figure PCTCN2022079096-appb-000014
在磷酸盐缓冲体系中，样品稳定性较差，在高温加速条件下发生剧烈降解，且不同浓度、不同pH条件下，降解率均较高。该结果表明样品不适宜在磷酸盐缓冲体系中长期存放。
在柠檬酸缓冲体系中，样品高温加速条件下稳定性相对较好，其聚体含量略高于磷酸盐体系样品，但降解含量远低于磷酸盐体系，总体而言，同等条件下，样品在柠檬酸缓冲液中稳定性高于在磷酸盐缓冲液中的稳定性。
根据本实施例结果，基本排除使用磷酸盐缓冲液作为样品的缓冲体系，后续在以柠檬酸缓冲体系为基础缓冲体系的前提上进行稳定性的研究。
2.2柠檬酸缓冲液浓度与pH范围筛选
选择柠檬酸缓冲溶液，对缓冲溶液浓度及pH进一步考察，以2个因素，3个水平进行DOE处方设计，pH选择5.5、6.0、6.5，缓冲溶液浓度为10mM、30mM、50mM，蛋白浓度均为25mg/mL。制剂样品在加速试验条件下进行考察，在50℃孵育1天后，测定蛋白纯度。
表6 制剂缓冲液确认试验50℃1天纯度检测结果
Figure PCTCN2022079096-appb-000015
结果：测定结果见表6。结果表明样品聚集程度受柠檬酸缓冲液浓度及pH的影响，在pH5.5-pH6.5范围内，聚体含量随pH的增加而增加，随柠檬酸缓冲液的浓度的增加而有所降低，表明该范围内相对较低的pH，相对较高浓度的柠檬酸缓冲液能够抑制重组融合蛋白聚集。
在降解程度方面，本实施例中样品的降解有随pH的增加而降低的趋势，但变化相对较小；柠檬酸缓冲液浓度变化对样品降解含量无明显影响。因此在本实施例的pH及浓度范围内，柠檬酸缓冲液浓度及pH对样品降解的影响较小
在单体含量方面，本实施例中样品单体含量随pH的增加而降低，其中pH小于等于6.0时，其含量相对稳定，变化较小，但pH大于6.0时，单体含量明显降低，表明pH大于6.0时，样品稳定性相对较差，而单体含量有随柠檬酸缓冲液浓度的增加而增加的趋势。
在本实施例中，通过磷酸盐缓冲液及柠檬酸缓冲液的对比实验，确定了柠檬酸缓冲液作为样品的基础缓冲液。进一步通过缓冲液浓度及pH范围确认试验，考察了柠檬酸缓冲液浓度及pH对样品的稳定性影响，从聚体，单体及降解三个方面进行分析，确认样品在pH5.5-pH6.5的柠檬酸缓冲液内稳定性均较好。
更优选的pH范围为pH5.5-pH6.0，更优选的缓冲液浓度为高浓度柠檬酸缓冲液，50mM柠檬酸最优。但是样品的最终产品作为皮下注射药物，需降低柠檬酸含量以减少注射刺激性。实验表明高浓度柠檬酸缓冲液对样品的稳定性效果优于低浓度柠檬酸缓冲液，考虑为高浓度柠檬酸缓冲液中盐离子浓度较高，从而增加了样品的稳定性，而盐离子浓度可在后续制剂中通过添加其他辅料而提高。因此可以将样品的缓冲液中柠檬酸浓度降低至10mM，以降低注射刺激性。
通过本实施例，确定了样品最优的缓冲体系为10mM柠檬酸缓冲液，pH5.5到pH6.0。
实施例3 制剂处方辅料筛选
在本实施例中，使用实施例2中确定的10mM pH5.5-6.0柠檬酸缓冲液，在缓冲液确定的情况下考察加入的辅料对蛋白稳定性的影响。从常用的对蛋白起稳定作用的四种辅料类型：糖醇类、盐类、表面活性剂类、氨基酸类中各取一种代表性辅料，进行DOE实验设计，通过高温加速试验快速考察样品的稳定性。
糖醇类选择蔗糖作为考察辅料，盐类选择NaCl作为考察辅料，表面活性剂选择聚山梨酯20作为考察辅料，氨基酸选择精氨酸作为考察辅料。蔗糖浓度范围为0-10％，NaCl浓度为0-140mM，精氨酸浓度范围为0-100mM，4因素两水平进行DOE设计，试验条件见下表7，其中序号9、10和11试验为中心重复。
表7：制剂处方辅料筛选DOE实验实施条件
Figure PCTCN2022079096-appb-000016
Figure PCTCN2022079096-appb-000017
按照上述表格条件制备相应体系样品，制备完成后进行50℃1天及2天高温加速试验，利用SE-HPLC法测样品纯度。
结果：测试结果如表8、表9所示。以表9中50℃孵育2天后纯度检测数据进行DOE统计学分析，该模型具有统计学意义，p＜0.05。50℃孵育2天后上述4种辅料对聚体的影响等值图见图1、图2。50℃孵育2天后上述4种辅料对降解的影响等值图见图3、图4。50℃孵育2天后上述4种辅料对单体的影响等值图见图5、图6。
表8 辅料筛选DOE实验50℃1天样品SEC结果
Figure PCTCN2022079096-appb-000018
表9：辅料筛选DOE实验50℃2天样品SEC结果
Figure PCTCN2022079096-appb-000019
Figure PCTCN2022079096-appb-000020
上表及图1的结果显示，氯化钠及蔗糖均对样品的聚集产生影响，在研究范围内，二者浓度越高，其对样品聚集的抑制效果越好。
聚山梨酯20浓度变化并未对聚体产生影响，而精氨酸的添加存在一定的聚集抑制效果，精氨酸含量越高，聚体含量越低。对表8中实验组1、2以及实验组3、4进行对比可见，在不含NaCl的制剂中，精氨酸起明显聚集抑制作用，而制剂中添加140mM NaCl后，添加精氨酸则未起到抑制聚集的效果，表明添加精氨酸只是起到增加盐离子浓度而增加样品稳定性的作用，其本身对样品无明显聚集抑制效果。
上表及图3的检测结果显示，蔗糖对样品的降解无明显影响，而氯化钠的添加使得降解有略微增加的趋势，但相对于聚体的变化，降解的变化极小，表明盐离子浓度的增加对降解无明显影响。
精氨酸的添加对降解有略微增加的趋势，其原因与氯化钠一致，精氨酸的添加增加了盐离子的浓度。而聚山梨酯20的添加使得降解有降低的趋势，表明聚山梨酯20具有少量抑制降解的效果。但考虑到生产工艺中添加聚山梨酯20的复杂性，聚山梨酯20在生产工艺中可不添加。
上表及图5的检测结果显示，蔗糖及氯化钠均对样品单体产生影响，二者含量越高，样品单体含量越高，根据前期聚体等值图(图1)分析结果，是由于蔗糖及氯化钠均有抑制样品聚集的效果，而对降解无明显影响，因此能够提高样品的纯度。
上表及图6的检测结果显示，精氨酸的添加有增加单体含量的趋势，这与其增加了盐离子浓度抑制了聚体有关，添加聚山梨酯20能少量增加单体含量，但增加量较少，表明聚山梨酯20对纯度的影响较小，从成本及工艺复杂程度出发，制剂中可不添加聚山梨酯20及同类的表面活性剂，最优的条件为添加0.05％的聚山梨酯20，能够进一步提高样品的稳定性。
在本实施例中，分别从聚体、降解及单体三个角度进行DOE数据分析，结果发现，主要起蛋白稳定性作用的为糖醇类及盐离子类辅料，这里主要指蔗糖及氯化钠， 但不限于这两种，表面活性剂的添加能够少量抑制样品的降解，包括但不限于聚山梨酯20。
实施例4 制剂处方筛选
4.1辅料含量确定
在本实施例中，在控制渗透压的情况下，考察不同含量辅料对样品稳定性的影响，确定辅料的含量。试验条件见表10。
表10 辅料含量对比实验条件
Figure PCTCN2022079096-appb-000021
结果：SEC检测结果见表11。
表11 辅料含量研究试验50℃1天样品SEC检测结果
Figure PCTCN2022079096-appb-000022
结果显示，不同含量的蔗糖和氯化钠均能提高样品稳定性，有效抑制降解。对比实验组1、2和3结果发现，pH对样品的降解存在一定的影响。
本实施例表明，在控制渗透压的前提下，蔗糖和氯化钠的不同含量及其组合均能有效提高样品的稳定性，均可作为样品的制剂处方辅料，但制剂处方的pH范围有待确定。
4.2制剂pH确定
以10mM柠檬酸缓冲液，100mM NaCl及3％蔗糖的制剂处方作为基础条件，考察不同pH条件下样品的变化趋势，进行50℃加速试验，试验条件及结果见表12。
表12 制剂处方pH研究及确认试验条件及SEC检测结果
Figure PCTCN2022079096-appb-000023
表12结果表明，在该制剂处方中，样品在pH5.2至pH6.4范围内稳定性均较好，50℃高温加速处理后样品聚体和降解的增加量均较小，符合样品长期保持的要求。
经实施例验证，该样品的优选制剂处方为柠檬酸缓冲液、糖醇类、盐离子类及表面活性剂类辅料几种成分的不同组合，pH为5.2至6.4。其中柠檬酸缓冲液浓度为10到50mM，糖醇类包括但不限于蔗糖，盐离子类包括但不限于氯化钠以及部分氨基酸，表面活性剂类包括但不限于聚山梨酯20。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种重组融合蛋白组合物，其特征在于，所述的组合物包括重组融合蛋白、缓冲液和稳定剂，
    其中，所述重组融合蛋白包括融合在一起的以下元件：(a)TNF受体或其活性片段；(b)OPG或其活性片段；和任选的(c)Fc片段。
  2. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述重组融合蛋白的浓度为2.5-50mg/mL，更佳地5-50mg/mL，更佳地10-40mg/mL，更佳地15-35mg/mL，更佳地20-25mg/mL，最佳地25mg/mL；
    所述缓冲液的浓度为1-100mM，较佳地为1-80mM，更佳地1-50mM，更佳地5-40mM，更佳地5-25mM，更佳地5-20mM，最佳地10mM；并且
    所述的稳定剂的含量为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％，以组合物的总重量计。
  3. 如权利要求1或2所述的重组融合蛋白组合物，其特征在于，所述的缓冲液选自磷酸盐缓冲液、柠檬酸缓冲液、或其组合；并且
    所述的稳定剂选自氨基酸、糖醇、无机盐，或其组合。
  4. 如权利要求3所述的重组融合蛋白组合物，其特征在于，所述的缓冲液选自下组：柠檬酸-磷酸缓冲液、柠檬酸-柠檬酸钠缓冲液、磷酸二氢钠-磷酸氢二钠缓冲液，或其组合；
    所述的氨基酸选自下组：精氨酸、甘氨酸、组氨酸，或其组合；
    所述的糖醇选自下组：蔗糖、甘露醇、海藻糖、麦芽糖、山梨醇，或其组合；和/或
    所述的无机盐选自下组：氯化钠、氯化钾、氯化钙、氯化镁，或其组合。
  5. 如权利要求3所述的重组融合蛋白组合物，其特征在于，所述的氨基酸(优选为精氨酸)的浓度为1-150mM，较佳地1-100mM，更佳地10-80mM，更佳地20-70mM，更佳地40-60mM，最佳地50mM；
    所述的糖醇(优选为蔗糖)的含量为0.5-15wt％，较佳地0.5-10wt％，更佳地1-8wt％，更佳地1.5-6wt％，更佳地2-5wt％，最佳地3wt％，以组合物的总重量计；和/或
    所述的无机盐(优选为氯化钠)的浓度为10-200mM，较佳地30-150mM，更佳 地60-140mM，更佳地70-130mM，更佳地90-110mM，最佳地100mM。
  6. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述的组合物的pH为5.0-7.0，较佳地5.0-6.5，更佳地5.2-6.4，更佳地5.3-6.3，最佳地5.4-6.0。
  7. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述的组合物进一步包括表面活性剂，所述的表面活性剂包括聚山梨酯20。
  8. 如权利要求7所述的重组融合蛋白组合物，其特征在于，所述表面活性剂的含量为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.005-0.5wt％，更佳地0.005-0.2wt％，更佳地0.008-0.15wt％，更佳地0.01-0.1wt％，更佳地0.02-0.08wt％，更佳地0.03-0.06wt％，最佳地0.05wt％，以组合物的总重量计。
  9. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述的组合物包括：
    Figure PCTCN2022079096-appb-100001
  10. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述的组合物包括：
    Figure PCTCN2022079096-appb-100002
  11. 如权利要求1所述的重组融合蛋白组合物，其特征在于，所述的组合物包括：
    Figure PCTCN2022079096-appb-100003
    Figure PCTCN2022079096-appb-100004
  12. 如权利要求1-11中任一项所述的重组融合蛋白组合物，其特征在于，所述的重组融合蛋白氨基酸序列如SEQ ID NO.:4所示。
  13. 一种如权利要求1-12中任一项所述的组合物或其制剂的用途，其特征在于，用于制备药物，所述药物用于(i)预防和/或治疗感染性疾病；和/或(ii)预防和/或治疗自身免疫性疾病；和/或(iii)预防和/或治疗骨质疏松或丢失；和/或(iv)预防和/或治疗肿瘤相关疾病。
  14. 如权利要求13所述的用途，其特征在于，所述肿瘤相关疾病选自下组：多发性骨髓瘤、骨相关的骨转移实体瘤、或其组合。
  15. 一种预防和/或治疗疾病的方法，包括步骤：给需要的对象施用施用如权利要求1所述的组合物或其制剂，从而治疗疾病，所述疾病选自下组：感染性疾病、自身免疫性疾病、骨质疏松或丢失、肿瘤相关疾病、或其组合。
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US20030077246A1 (en) * 1999-12-16 2003-04-24 Welcher Andrew A. TNFr/OPG-like molecules and uses thereof
US20110152173A1 (en) * 2008-07-02 2011-06-23 Emergent Product Development Seattle ,LLC TNF-a ANTAGONIST MULTI-TARGET BINDING PROTEINS
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