WO2022033480A1 - 一种液体制剂及其应用 - Google Patents

一种液体制剂及其应用 Download PDF

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Publication number
WO2022033480A1
WO2022033480A1 PCT/CN2021/111804 CN2021111804W WO2022033480A1 WO 2022033480 A1 WO2022033480 A1 WO 2022033480A1 CN 2021111804 W CN2021111804 W CN 2021111804W WO 2022033480 A1 WO2022033480 A1 WO 2022033480A1
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liquid formulation
buffer
concentration
liquid
fusion protein
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PCT/CN2021/111804
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English (en)
French (fr)
Inventor
陶松树
徐卫涛
马海立
余宇婷
徐临凤
刘恒
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隆延生物科技（上海）有限公司
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Priority to CN202180058922.6A priority Critical patent/CN116249552A/zh
Publication of WO2022033480A1 publication Critical patent/WO2022033480A1/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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]

Definitions

  • the present application relates to the field of biomedicine, in particular to a liquid preparation of recombinant hEPO-Fc fusion protein.
  • Proteins are large and complex molecules that need to be in their native conformation to remain biologically active. Liquid formulations are the primary choice in the preparation of therapeutic proteins or pharmaceutical compositions thereof. However, proteins are prone to aggregation, degradation, denaturation or chemical modification over time during manufacture or storage, losing biological activity.
  • protein molecules are linked covalently or non-covalently to form soluble high molecular weight protein aggregates, or insoluble protein aggregates that elicit undesired immune responses in patients after administration.
  • the formation of protein aggregates can clog the syringe or pump during use, creating a safety hazard for the patient.
  • the present application provides a liquid preparation, which may include recombinant hEPO-Fc fusion protein, buffer and stabilizer, and may also include surfactant, or pH adjuster.
  • the liquid preparation has stability, and under different placement conditions, such as light, high temperature, shaking and/or freeze-thaw conditions, the recombinant hEPO-Fc fusion protein forms protein aggregates or fragments in the preparation.
  • the liquid preparation has one or more of the following properties: (1) can be stored at 37° C. for at least 2 weeks, and wherein the aggregate increase of the recombinant hEPO-Fc fusion protein is less than 10%; (2) at 37° C.
  • the application provides a liquid formulation comprising a recombinant hEPO-Fc fusion protein, a buffer and a stabilizer, wherein the recombinant hEPO-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, and The concentration of the recombinant hEPO-Fc fusion protein is 0.1 mg/ml-10 mg/ml.
  • the buffer is selected from one or more of the group consisting of phosphate buffer, Tris buffer, and histidine hydrochloride buffer (His/HCl).
  • the buffer is selected from one or more of the group consisting of phosphate buffer and Tris buffer.
  • the buffer is a phosphate buffer.
  • the concentration of the buffer is about 1 mM to 50 mM. In certain embodiments, the concentration of the buffer is about 1 mM to 10 mM. In certain embodiments, the concentration of the buffer is about 10 mM.
  • the stabilizer includes arginine or a pharmaceutically acceptable salt thereof. In certain embodiments, the stabilizer is arginine hydrochloride.
  • the concentration of the stabilizer is from about 10 mM to 500 mM. In certain embodiments, the concentration of the stabilizer is about 10 mM to 150 mM. In certain embodiments, the concentration of the stabilizer is about 150 mM.
  • the liquid formulation includes a surfactant.
  • the surfactant includes polysorbate.
  • the surfactant includes polysorbate 20 and/or polysorbate 80.
  • the polysorbate 20 is present in an amount of about 0.0001%-0.1% (w/w). In certain embodiments, the polysorbate 20 is present in an amount of about 0.001%-0.01% (w/w). In certain embodiments, the polysorbate 20 is present in an amount of about 0.001%-0.05% (w/w). In certain embodiments, the polysorbate 20 is present in an amount of about 0.01%-0.1% (w/w). In certain embodiments, the polysorbate 20 is present in an amount of about 0.001%-0.01% (w/w). In certain embodiments, the polysorbate 20 is present at about 0.01% (w/w).
  • the stabilizer further comprises a saccharide comprising one or more selected from the group consisting of sucrose, trehalose, and cyclodextrin.
  • the carbohydrates include one or more selected from the group consisting of sucrose and trehalose.
  • the carbohydrate is present in an amount of about 1% to 50%. In certain embodiments, the carbohydrate is present in an amount of about 2% to 5%.
  • the liquid formulation further comprises a chelating agent.
  • the chelating agent comprises EDTA.
  • the EDTA is at a concentration of about 0.001 mM to 20 mM. In certain embodiments, the EDTA is at a concentration of about 5 mM to 10 mM.
  • the liquid formulation may include a pH adjusting agent such that the pH of the liquid formulation is about 6.0-9.0. In certain embodiments, the pH of the liquid formulation is about 6.5-8.5. In certain embodiments, the pH of the liquid formulation is about 7-8.
  • the liquid formulation has one or more of the following properties:
  • (1) can be stored at 37°C for at least 2 weeks, and wherein the aggregate increase of the recombinant hEPO-Fc fusion protein is less than 10%;
  • (2) can be stored at 37°C for at least 2 weeks, and wherein the fragment increase of the recombinant hEPO-Fc fusion protein is less than 8%;
  • the present application provides the use of the liquid preparation in the preparation of a medicament for preventing, relieving and/or treating anemia.
  • the present application provides the liquid formulation for preventing, alleviating and/or treating anemia.
  • the present application provides a method of preventing, alleviating and/or treating anemia, comprising using the liquid formulation.
  • Figure 1 shows the SE-HPLC results of the stability of liquid formulations with different buffers under different storage conditions.
  • Figure 2 shows the rCE-SDS test results of the stability of liquid preparations with different buffers under different storage conditions.
  • Figure 3 shows the nrCE-SDS test results of the stability of liquid preparations with different buffers under different storage conditions.
  • Figure 4 shows the nrCE-SDS detection results of the stability of liquid preparations with different buffers under different storage conditions, where A: low molecular weight fragment (LMWS) content, B: high molecular weight polymer (HMWS) content, C: 28min fragment content, D: 24min fragment content.
  • LMWS low molecular weight fragment
  • HMWS high molecular weight polymer
  • Figure 5 shows the results of the icIE assay for the stability of liquid formulations with different buffers under shaking conditions.
  • Figure 6 shows the results of the icIE assay for the stability of liquid formulations with different buffers under light conditions.
  • Figure 7 shows the icIE test results of the stability of liquid formulations with different buffers at 37C/2W.
  • Figure 8 shows the SE-HPLC detection results of liquid formulations with different excipients under control (A), 25C/2W (B) and freeze-thaw (C) conditions.
  • Figure 9 shows the high molecular weight polymer content of liquid formulations with different excipients at 37/2W.
  • Figure 10 shows the SE-HPLC test results of the stability of liquid formulations with different excipients added to the same buffer system.
  • Figure 11 shows the SE-HPLC test results of the stability of liquid formulations with different excipients under different conditions.
  • Figure 12 shows the HMWS content of liquid formulations with different excipients under different conditions.
  • Figure 13 shows the LMWS content of liquid formulations with different excipients under different conditions
  • Figure 14 shows the particle size of insoluble particles in liquid formulations with PS20 or cyclodextrin added under different conditions.
  • Figure 15 shows the particle size of insoluble particles of liquid formulations with NaCl or arginine hydrochloride added under different conditions.
  • Figure 16 shows the particle size of insoluble particles of liquid formulations of different types and concentrations of surfactants, where SK: shaking at room temperature; RT: standing at room temperature.
  • Figure 17 shows the main peak content of liquid formulations detected by SE-HPLC at different concentrations of phosphate buffer.
  • Figure 18 shows the HMWS content of liquid formulations detected by SE-HPLC at different concentrations of phosphate buffer.
  • Figure 19 shows the LMWS content of liquid formulations detected by SE-HPLC at different concentrations of phosphate buffer.
  • Figure 20 shows the main peak content of liquid preparations detected by SE-HPLC at different concentrations of Arg-HCl.
  • Figure 21 shows the HMWS content of liquid formulations detected by SE-HPLC at different concentrations of Arg-HCl.
  • Figure 22 shows the LMWS content of liquid formulations detected by SE-HPLC at different concentrations of Arg-HCl.
  • hEPO-Fc fusion protein generally refers to a fusion protein formed by the fusion of human erythropoietin (hEPO) and the Fc fragment of human IgG.
  • Erythropoietin (EPO) is an approximately 30.4 kilodalton glycoprotein hormone that promotes the proliferation of erythroid progenitor cells and maintains their differentiation into mature erythrocytes.
  • Recombinant human EPO rHuEPO
  • the IgG may be IgG2.
  • the hEPO can be indirectly fused to the Fc fragment of human IgG through a peptide linker.
  • Two hEPO-IgG2 Fc fusion proteins in the recombinant hEPO-Fc fusion proteins were combined in the IgG2 Fc region via disulfide bonds, resulting in an EPO dimer linked to the N-terminus of the Fc fragment.
  • Recombinant hEPO-Fc fusion proteins may contain glycosylation sites and disulfide bonds.
  • the recombinant hEPO-Fc protein may comprise the following amino acid sequence:
  • the terms “stability” and “stable” generally refer to a liquid formulation comprising a protein (eg, recombinant hEPO-Fc fusion protein), the protein (eg, recombinant hEPO-Fc fusion protein) in a given No, or only minimal aggregation, degradation or fragmentation occurs under the conditions of manufacture, preparation, transport and/or storage.
  • a “stable” liquid formulation retains biological activity under given conditions of manufacture, preparation, transportation and/or storage.
  • SE-HPLC size exclusion high performance liquid chromatography
  • rCE-SDS reducing capillary electrophoresis
  • nrCE-SDS non-reducing capillary electrophoresis
  • icIEF full-column imaging capillary isoelectric focusing electrophoresis
  • PM particle size detection techniques to measure the degree of aggregation, degradation or fragmentation, etc. of the formulation to assess the stability of the protein (eg, recombinant hEPO-Fc fusion protein).
  • proteins with a molecular weight lower than the recombinant hEPO-Fc fusion protein can be regarded as fragments, and proteins with a molecular weight higher than the recombinant hEPO-Fc fusion protein can be regarded as aggregates.
  • saccharide may generally contain varying amounts of sugar (saccharide) units, such as monosaccharides, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides and polysaccharides.
  • Carbohydrates also include their derivatives, such as amino sugars, sugar alcohols, aldoses, and the like. Examples of carbohydrates may include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, sucrose, trehalose, lactose, maltose, maltodextrin, dextran, cyclodextrin and/or starch.
  • polysorbate generally refers to the oleic acid esters of sorbitol and its anhydrides, eg, copolymerized with ethylene oxide.
  • Polysorbates may include polysorbate 20 (polyethylene oxide (20) sorbitan monolaurate, Tween 20, or PS20), and polysorbate 80 (polyethylene oxide (80) Sorbitan monolaurate, Tween 80 or PS80).
  • anemia generally refers to a broadly defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood.
  • anemia can be caused by a primary disorder in the production or survival of red blood cells. More commonly, anemia is secondary to diseases of other systems (Weatherall & Provan (2000) Lancet 355, 1169-1175).
  • Anemia can be caused by a decreased rate of red blood cell production or increased rate of destruction, or by loss of red blood cells due to bleeding.
  • Anemia can be caused by a variety of conditions including, for example, chronic renal failure, chemotherapy treatment, myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.
  • the anemia may be caused by an ineffective erythropoietic disorder.
  • w/w generally refers to “mass to volume ratio”, which is the ratio of the mass of the components to the volume of the formulation.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides a liquid formulation comprising a recombinant hEPO-Fc fusion protein, and the recombinant hEPO-Fc fusion protein may comprise the amino acid sequence shown below:
  • the recombinant hEPO-Fc fusion protein may comprise at least 70% (eg, at least 75%, 80%, 85%, 90%, 95%, 96%, 97%) of the amino acid sequence set forth in SEQ ID NO: 1 , 98%, 99% or more) amino acid sequences of sequence identity.
  • the concentration of the recombinant hEPO-Fc fusion protein can be about 0.1 mg/ml-10 mg/ml, for example, the concentration of the recombinant hEPO-Fc fusion protein can be about 0.1 mg/ml-9 mg/ml ml, about 0.1mg/ml-8mg/ml, about 0.1mg/ml-7mg/ml, about 0.1mg/ml-6mg/ml, about 0.1mg/ml-5mg/ml, about 0.1mg/ml-4mg/ml ml, about 0.5 mg/ml-4 mg/ml, about 0.5 mg/ml-3 mg/ml, or about 0.5 mg/ml-2 mg/ml.
  • the concentration of the recombinant hEPO-Fc fusion protein can be about 2 mg/ml.
  • the recombinant hEPO-Fc fusion protein may comprise a protein dimer composed of two of the above amino acid sequences.
  • the liquid formulation may comprise a buffer
  • the buffer may be selected from one or more of the following group: phosphate buffer, Tris buffer, and histidine hydrochloride buffer (His/HCl ).
  • the formulation contains a buffer or a buffer system, it also means that the formulation contains a buffer, and a buffer system is formed in the formulation through the buffer.
  • the effect of different buffer systems such as: phosphate buffer, Tris buffer, sodium acetate buffer and histidine hydrochloride buffer (His/HCl) on the recombinant hEPO-Fc fusion protein was evaluated impact on stability.
  • the buffer may be selected from one or more of the group consisting of phosphate buffer and Tris buffer.
  • the buffer may be a phosphate buffer.
  • the phosphate buffer may include sodium phosphate buffer (which may include sodium dihydrogen phosphate, disodium hydrogen phosphate), potassium phosphate buffer, or a combination of both.
  • the buffer may be Tris buffer.
  • the buffer may comprise a cross-buffer consisting of two or more buffers selected from the group consisting of phosphate buffer, Tris buffer, and histidine hydrochloride buffer ( His/HCl).
  • the buffer may be a His/Tris buffer.
  • the buffering agent can play the role of buffering external pH changes, so as to keep the pH value of the solution within a certain range.
  • the buffer in the liquid formulation is formed of a buffer, and the concentration of the buffer may be about 1 mM-50 mM.
  • the concentration of the buffer may be about 1 mM-45 mM, about 1 mM-40 mM, about 1 mM-35 mM, about 1 mM-30 mM, about 1 mM-25 mM, about 1 mM-20 mM, about 1 mM-15 mM, about 2 mM-15 mM, About 2mM-20mM or about 5mM-20mM.
  • the concentration of the buffer in the liquid formulation may be about 10 mM.
  • the liquid formulation may further include a pH adjusting agent, which may cause the pH of the liquid formulation to be about 6.0-9.0, eg, about 6.5-8.5, about 6.5-8.0, or is about 6.5-7.5.
  • the pH of the liquid formulation may be 7.0-8.0.
  • the pH of the liquid formulation may be about 6.5, about 7.0, about 7.5, about 8.0 or about 8.5.
  • the pH of the liquid formulation may be about 7.0.
  • the pH adjusting agent may be an organic or inorganic acid, a base, a neutralizing agent and/or a buffering agent.
  • the liquid formulations may also contain one or more suitable stabilizers, which may be pharmaceutically acceptable excipients, to protect the pharmaceutically active ingredient during manufacture, storage and/or use (eg, the recombinant hEPO-Fc fusion protein) is immune to chemical and/or physical degradation. Due to the large molecular weight of proteins, the degradation of amino acids is also more frequent, and various physical and chemical effects can cause changes in amino acids such as deamination, cyclization, and chemical bond breakage.
  • suitable stabilizers include, but are not limited to, suitable carbohydrates, amino acids, polyols, or suitable derivatives or mixtures thereof.
  • Suitable amino acids that can be used as stabilizers include, but are not limited to, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, benzene Alanine, tyrosine, tryptophan, methionine, serine, proline, cysteine and/or cystine, or any combination of the above and pharmaceutically acceptable salts thereof.
  • the amino acid may exist in the form of an amino acid salt, eg, an amino acid hydrochloride.
  • suitable amino acids may include arginine, or the arginine may be added to the liquid formulation in the form of arginine hydrochloride.
  • the concentration of the amino acid may be about 10 mM to 500 mM.
  • the concentration of the amino acid can be about 10 mM-450 mM, about 10 mM-400 mM, about 10 mM-350 mM, about 10 mM-300 mM, about 50 mM-300 mM, about 50 mM-250 mM, about 50 mM-200 mM, or about 100 mM-200 mM.
  • the concentration of arginine or a pharmaceutically acceptable salt thereof (eg, arginine hydrochloride) in the liquid formulations described herein is about 10-500 mM.
  • the concentration of arginine or a pharmaceutically acceptable salt thereof (e.g., arginine hydrochloride) in the liquid formulations described herein is about 150 mM.
  • stabilizers that can be used as the liquid formulation may include sugars.
  • Suitable saccharides may include, but are not limited to, amino sugars, sugar alcohols, aldoses, and the like.
  • carbohydrates may include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, sucrose, trehalose, lactose, maltose, maltodextrin, dextran, cyclodextrin, and starch, or suitable mixture.
  • the carbohydrate may include one or more selected from the group consisting of sucrose, trehalose, and cyclodextrin.
  • the carbohydrates may include one or more selected from the group consisting of sucrose and trehalose.
  • the carbohydrate can include sucrose.
  • the content of the carbohydrate may be about 1%-50%, for example, about 1%-50%, about 1%-45%, about 1%-40%, about 1%-35%, About 1%-30%, about 1%-25%, about 1%-20%, about 1%-15%, about 1%-10%.
  • the liquid formulation may also include a surfactant.
  • the surfactant can be a pharmaceutically acceptable excipient for protecting a protein (eg, the recombinant hEPO-Fc fusion protein) from various intensifying conditions, such as stirring, shearing, exposure to high temperature, and the like.
  • the surfactant can also solubilize, or prevent the protein from polymerizing during shaking, adsorption or precipitation when in contact with the inner packaging material, air, or rubber stopper.
  • the surfactants may include, but are not limited to, polysorbates (Tween, or polyoxyethylene sorbitan fatty acid esters), polyoxyethylene alkyl ethers (eg, Brij), alkyl phenyl polyoxyethylene ethers ( For example, Triton-X), polyoxyethylene-polyoxypropylene copolymers (eg, poloxamers, pluronics), sodium dodecyl sulfonate (SDS), and any combination of the foregoing.
  • the surfactant may include polysorbate.
  • the surfactant may include polysorbate 20 and/or polysorbate 80.
  • the content of the surfactant may be about 0.0001%-0.1% (w/w), for example, the content of the surfactant may be about 0.0005%-0.1% (w/w), about 0.001%-0.1% (w/w), about 0.001%-0.09% (w/w), about 0.001%-0.08% (w/w), about 0.001%-0.07% (w/w), about 0.001% -0.06% (w/w), about 0.001%-0.05% (w/w), about 0.002%-0.04% (w/w), about 0.003%-0.03% (w/w), about 0.004%-0.025 % (w/w), about 0.001%-0.045% (w/w), about 0.001%-0.04% (w/w), about 0.001%-0.035% (w/w), about 0.001%-0.03% ( w/w).
  • the content of the polysorbate 20 may be 0.001%-0.05% (w/w).
  • the surfactant in the liquid formulation may include polysorbate 20 and/or polysorbate 80 at a concentration of about 0.001% to about 0.1% (w/w).
  • the liquid formulation may further comprise a chelating agent.
  • Chelating agents generally refer to multifunctional molecules with a number of negatively charged ligands and/or multielectron ligands that can sequester metal ions with varying affinities. Suitable multi-electron functional groups include carboxyl, hydroxyl and amino groups.
  • chelating agents include aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-ylidene Amino diacetic acid (ADA), bis(aminoethyl) glycol ether-N,N,N',N'-tetraacetic acid (EGTA), trans-cyclohexanediaminetetraacetic acid (DCTA), glutamic acid and/or aspartic acid, hydroxyaminocarboxylic acids such as N-hydroxyethyliminodiacetic acid (HIMDA), N,N-di-hydroxyethylglycine (N-bis(hydroxyethyl)glycine) and/or N-(trimethylolmethyl)glycine (N-tris(hydroxymethyl)methylglycine), and N-substituted glycines such as N-sub
  • the chelating agent may comprise EDTA.
  • the concentration of EDTA can be about 0.001 mM to 20 mM, eg, about 0.001 mM to 18 mM, about 0.001 mM to 16 mM, about 0.001 mM to 14 mM, about 0.001 mM to 12 mM, about 0.001 mM to 10 mM, about 1 mM to 1 mM 14 mM, about 1 mM to 12 mM, or about 1 mM to 10 mM.
  • the liquid preparation may include the following substances: the recombinant hEPO-Fc fusion protein, the buffer and the stabilizer.
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 0.1 mg/ml to 10 mg/ml, phosphate buffered saline (PB) at a concentration of about 1 mM to 50 mM, and a concentration of about 10 mM to 500 mM arginine (or arginine hydrochloride), may also include about 0.0001%-0.1% (w/w) polysorbate 20 or polysorbate 80, and/or about 0.001 mM to 20 mM EDTA, And the pH can be about 6.0-9.0.
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 1-5 mg/ml, phosphate buffered saline (PB) at a concentration of about 1-20 mM, arginine at a concentration of about 50 mM-300 mM acid (or arginine hydrochloride) and about 0.001%-0.02% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-8.5.
  • PB phosphate buffered saline
  • arginine at a concentration of about 50 mM-300 mM acid (or arginine hydrochloride)
  • w/w polysorbate 20 or polysorbate 80
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at about 10 mM, and arginine (or arginine at a concentration of about 150 mM) hydrochloride), and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine or arginine at a concentration of about 150 mM hydrochloride
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at about 10 mM, arginine (or arginine at a concentration of about 150 mM) hydrochloride) and about 0.005%-0.01% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine or arginine at a concentration of about 150 mM hydrochloride
  • w/w polysorbate 20 or polysorbate 80
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at a concentration of about 1-50 mM, arginine at a concentration of about 10-500 mM (or arginine hydrochloride) and about 0.001%-0.1% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine at a concentration of about 10-500 mM (or arginine hydrochloride)
  • w/w polysorbate 20 or polysorbate 80
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at about 1 mM, arginine (or arginine hydrochloride) and about 0.001%-0.1% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine or arginine hydrochloride
  • polysorbate 20 or polysorbate 80 the pH can be adjusted to about 6.5-7.5.
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at a concentration of about 1-10 mM, arginine at a concentration of about 10-500 mM (or arginine hydrochloride) and about 0.001%-0.1% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine at a concentration of about 10-500 mM (or arginine hydrochloride)
  • w/w polysorbate 20 or polysorbate 80
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at 10 mM, arginine (or arginine at a concentration of about 150 mM) hydrochloride) and about 0.01% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • arginine or arginine at a concentration of about 150 mM hydrochloride
  • polysorbate 20 or polysorbate 80 the pH can be adjusted to about 6.5-7.5.
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 1-2 mg/ml, phosphate buffered saline (PB) at a concentration of about 1-10 mM, spermatozoa at a concentration of about 50-150 mM amino acid (or arginine hydrochloride) and about 0.01%-0.1% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6.5-7.5.
  • PB phosphate buffered saline
  • spermatozoa at a concentration of about 50-150 mM amino acid (or arginine hydrochloride)
  • w/w polysorbate 20 or polysorbate 80
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, phosphate buffered saline (PB) at a concentration of 1-10 mM, arginine at a concentration of about 50-150 mM ( or arginine hydrochloride) and about 0.01% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 7.
  • PB phosphate buffered saline
  • arginine at a concentration of about 50-150 mM ( or arginine hydrochloride)
  • 0.01% (w/w) polysorbate 20 or polysorbate 80 the pH can be adjusted to about 7.
  • the liquid formulation may include the following: the recombinant hEPO-Fc fusion protein at a concentration of about 2 mg/ml, Tris at about 10 mM, arginine (or arginine hydrochloride) at a concentration of about 150 mM, and about 0.005%-0.01% (w/w) polysorbate 20 or polysorbate 80, and the pH can be adjusted to about 6-8.
  • the present application provides a liquid formulation that is stable and has a low content of protein aggregates or fragments placed in the formulation.
  • stable formulations are those that retain their physical stability, chemical stability, and/or biological activity after storage for a certain period of time.
  • the stability of the liquid preparation can be evaluated by the content of protein (eg, recombinant hEPO-Fc fusion protein), the content of protein fragments and/or the content of protein aggregates therein.
  • SE-HPLC size exclusion high performance liquid chromatography
  • rCE-SDS reducing capillary electrophoresis
  • nrCE-SDS non-reducing capillary electrophoresis
  • icIEF full-column imaging capillary isoelectric focusing
  • PM particle size detection techniques measure the protein, fragment and/or aggregate content of interest in the formulation, or the particle size of insoluble matter, to assess the protein (e.g., recombinant hEPO- Fc fusion protein).
  • the liquid formulation has one or more of the following properties:
  • (1) Can be stored at 37°C for at least 2 weeks, and wherein the recombinant hEPO-Fc fusion protein has an aggregate increase of less than 10%, eg, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% or less;
  • fragment increase of the recombinant hEPO-Fc fusion protein is less than 8%, eg, less than 7%, less than 6%, less than 5%, less than 4% %, less than 3%, less than 2%, less than 1% or less;
  • the recombinant hEPO-Fc fusion protein is at least 90% pure, eg, at least 91%, at least 92%, at least 93%, at least 94% pure , at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher;
  • the purity of the recombinant hEPO-Fc fusion protein is at least 95%, for example, at least 96%, at least 97%, at least 98%, At least 99% or higher.
  • the present application provides the use of the liquid preparation in the preparation of a medicament for preventing, relieving and/or treating anemia.
  • the present application provides the liquid formulation for preventing, alleviating and/or treating anemia.
  • the present application provides a method of preventing, alleviating and/or treating anemia, comprising using the liquid formulation.
  • the liquid formulation can be used for the treatment of thalassemia syndrome and also for the treatment of ineffective erythropoiesis disorders.
  • Such disorders include sideroblastic anemia (hereditary or acquired); erythropoietic anemia (types I and II); sickle cell anemia; hereditary spherocytosis; pyruvate kinase deficiency; Megaloblastic anemia, which may be caused by conditions: such as folate deficiency (due to congenital disease, decreased intake or increased demand), cobalamin deficiency (due to congenital disease, pernicious anemia, impaired absorption, pancreatic insufficiency, or reduced intake), certain drugs, or of unknown etiology (congenital dyserythropoietic anemia, refractory megaloblastic anemia, or erythroleukemia); anemias of myelopathy, including myelofibrosis (myeloid metaplasia) and bone marrow tub
  • the liquid preparations prepared in each example were sterilized by filtration with a 0.22 ⁇ m filter device (Millipore TM , SLGV004SL), and aseptically filled into sterilized glass bottles (Schott Xinkang Pharmaceutical Packaging Co., Ltd., China Boron 276) for sealing. After storing the glass bottle containing the liquid preparation under each of the following conditions, the test was performed using the above method.
  • Control conditions 5°C, standing for 14 days. Shaking conditions: 200rpm horizontal centrifugation for 14 days. Freeze-thaw conditions: -70°C to 5°C with 5 slow cycles. Lighting conditions: 4500Lux for 14 days, UV irradiation for 5 days.
  • 25C/1W condition stand at 25°C for 1 week.
  • 25C/2W condition 2 weeks at 25°C 25C/1M condition: 1 month at 25°C.
  • 25C/2M conditions stand at 25°C for 2 months.
  • 25C/3M conditions stand at 25°C for 3 months.
  • 37C/1W condition stand at 37°C for 1 week.
  • 37C/2W condition stand at 37°C for 2 weeks.
  • 37C/1M conditions stand at 37°C for 1 month.
  • 37C/2M conditions stand at 37°C for 2 months.
  • 37C/3M conditions stand at 37°C for 3 months.
  • Appearance detection After placing samples of different formulations under different conditions, the clarity was observed with naked eyes.
  • Size exclusion high performance liquid chromatography (SE-HPLC) detection method Agilent high performance liquid chromatography 1260, UV/visible light detector, chromatographic column TSKgel G3000SW XL (7.8x 300mm, 5 ⁇ m, TOSOH), mobile phase 40mM PB+0.3 M Na 2 SO4, pH 6.5, flow rate 0.6 mL/min, sample volume 20 ⁇ g, sample chamber temperature 2-8 °C, detection time 35 min, absorption wavelength 280 nm, record chromatogram, and calculate LP by area normalization after integration -001 main peak, relative content of high molecular weight components and low molecular weight components.
  • SE-HPLC Size exclusion high performance liquid chromatography
  • the LP-001 protein was changed to 10 mM phosphate buffer (pH 7.0), and the protein concentration was adjusted to 5 mg/mL. 20 ⁇ L of 5mg/mL LP-001 protein was mixed with 50 ⁇ l of 1% SDS sample buffer (100mM Tirs-HCl, pH 9.0), then 5 ⁇ L of reducing agent ( ⁇ -mercaptoethanol) and 25 ⁇ L of ultrapure water were added, and incubated at 70°C After 10 mins, it was cooled, transferred to a sample injection vial, placed in a 15 °C sample injection tray of an Agilent G7100A instrument, and an uncoated capillary (inner diameter of 50 ⁇ m, effective separation length of 8.5 cm) was used to detect the absorbance value at a wavelength of 220 nm.
  • the relative contents of the main peak, high molecular weight components and low molecular weight components of LP-001 were determined according to the percentage of corrected peak area by integrating the electropherogram and calculating according to the area normalization method.
  • Non-reducing capillary electrophoresis (nrCE-SDS) detection method :
  • the LP-001 protein was changed to 10 mM phosphate buffer (pH 7.0), and the protein concentration was adjusted to 5 mg/mL. 20 ⁇ L of 5mg/mL LP-001 protein was mixed with 50 ⁇ L of 1% sample buffer (100mM Tirs-HCl, pH 9.0), then 5 ⁇ L of alkylation reagent (1M iodoacetic acid) and 25 ⁇ L of ultrapure water were added, and incubated at 70°C After 10 mins, it was cooled, transferred to a sample injection vial, placed in a 15 °C sample injection tray of an Agilent G7100A instrument, and an uncoated capillary (inner diameter of 50 ⁇ m, effective separation length of 8.5 cm) was used to detect the absorbance value at a wavelength of 220 nm.
  • the relative contents of the main peak, high molecular weight components and low molecular weight components of LP-001 were determined according to the percentage of corrected peak area by integrating the electropherogram and calculating according to the area normalization method.
  • LP-001 protein was mixed with final concentration of 0.35% methylcellulose, 4% 3-10 ampholyte, 0.5% pI Marker (4.22, 9.46) and 4M urea, using Maurice C. and fluorocarbon coating from Protein Simple Company. Layer capillary, focusing at 1500V for 1min and 3000V for 10mins, respectively, at a wavelength of 280nm to detect the charge variants of LP-001 protein. According to the area normalization method, the relative contents of the main peak components, acidic peak components and basic peak components are reported
  • the arginine was all added to the liquid formulation in the form of arginine hydrochloride.
  • Liquid formulations were formulated according to the formulation in Table 1 below, pH adjusted.
  • HMWS high molecular weight components
  • LMWS low molecular weight components
  • FIG. 3 shows the results of nrCE-SDS in Fig. 3 and Fig. 4 .
  • Figure 4C and Figure 4D show that LMWS mainly concentrates on the fragment peaks at 28min and 24min (the main peak is 32min); according to the peak time, it is speculated that 24min may correspond to a single-chain peak.
  • Liquid formulations were formulated according to the formulation in Table 5 below, and the pH was adjusted.
  • each group was placed under different placement conditions for a period of time before detection.
  • the groups with pH 6.0, 7.0 and 8.0 in the PB buffer system are called PB6, PB7 and PB8, respectively
  • the group with pH 8.0 in the Tris buffer system is called Tris8.
  • Figures 8A-8C show the percentage of main peaks detected by SE-HPLC in each group under control (A), 25C/2W (B), and freeze-thaw (C) conditions, respectively.
  • Figure 10 shows the detection results of SE-HPLC in the PB7 system with different stabilizers added and placed under different conditions.
  • the results showed that the percentage of the main peak in the group with arginine hydrochloride, sorbitol, sucrose and trehalose was higher than that in the control group without stabilizer, and the percentage of the main peak in the group with glycine was lower than that in the control group, indicating that arginine hydrochloride Salt, sorbitol, sucrose and trehalose have good stabilizing effect on protein, and not any stabilizer can stabilize protein, but glycine is not conducive to stabilization.
  • the liquid formulation was formulated according to the formulation in Table 6 below, pH adjusted.
  • each group was placed under different placement conditions for a period of time before detection.
  • the groups with pH 6.0, 7.0 and 8.0 in the PB buffer system are called PB6, PB7 and PB8 respectively
  • the group with pH 8.0 in the Tris buffer system is called Tris8.
  • Figure 14 shows group 3 and group 5, comparing the PM results of group 3 and group 5 added with PS20, group 5 with cyclodextrin added, after a certain period of time under each condition, group 5 produced larger molecular size , indicating that the protein formed aggregates, while group 3 containing arginine hydrochloride and PS-20 was stable and not easy to form aggregates (Fig. 14).
  • Figure 15 shows the PM results of group 1 with added sodium chloride and group 3 with arginine hydrochloride under different placement conditions.
  • Liquid formulations were formulated according to the following Table 8 formulation, pH adjusted.
  • each group was placed under different placement conditions for a period of time before detection.
  • the groups with pH 6.0, 7.0 and 8.0 in the PB buffer system are called PB6, PB7 and PB8 respectively
  • the group with pH 8.0 in the Tris buffer system is called Tris8.
  • SE-HPLC detects the protein content and the content of the generated aggregates or fragments, and the results are shown in Table 9 below. All groups were placed at 25°C for 2 weeks, and the liquid preparations were stable, and at least 95% of the protein molecules would not be degraded into fragments or form aggregates; placed at 37°C for 2 weeks, the liquid preparations were also stable, with at least 90 % of the protein molecules will not be degraded into fragments or form aggregates, the amount of fragments generated does not exceed 4.0%, and the amount of aggregates generated does not exceed 7%.
  • nrCE-SDS detects the protein content and the content of the generated aggregates or fragments, and the results are shown in Table 10 below. All groups were placed at 25°C or 37°C for 2 weeks, and the liquid formulations were stable, and at least 95% of the protein molecules did not degrade into fragments or form aggregates. Fragment generation did not exceed 5% even after 2 weeks of storage at 37°C.
  • ND means that no results can be detected under the detection conditions.
  • ND means that no results can be detected under the detection conditions.
  • Example 6 Effects of different concentrations of phosphate buffer and excipients (arginine hydrochloride) on the stability of liquid formulations
  • LP-001 at 2 mg/mL was exchanged into the formulations below, and the concentrations of phosphate buffer (1-50 mM) and arginine hydrochloride (Arg-HCl, 10-500 mM) were studied.

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Abstract

一种液体制剂，其包含重组hEPO-Fc融合蛋白、缓冲液和稳定剂。所述液体制剂还可包含其他辅料。还提供了所述液体制剂在制备治疗贫血药物中的用途。

Description

一种液体制剂及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种重组hEPO-Fc融合蛋白的液体制剂。
背景技术
蛋白质是大而复杂的分子，需要处于其天然构象以保持生物学活性。液体制剂是在制备治疗用蛋白质或其药物组合物的主要选择。然而，在制造或存储过程中，随着时间蛋白质易于发生聚集、降解、变性或化学修饰，而损失生物学活性。例如，蛋白质分子以共价键或非共价键连接，形成可溶性的高分子量蛋白质聚集体，或不溶性的蛋白质聚集体，给药后在患者中引起不期望的免疫反应。此外，蛋白质聚体的形成还会在使用期间阻塞注射器或泵，对患者造成安全隐患。
因此，为了实现治疗性蛋白质的临床效价，需要稳定的用于蛋白质制备或存储的新制剂，其包含这种治疗性蛋白质，以使蛋白质在制造过程中和/或在存储、运输、使用过程中不会造成显著的蛋白质聚体或片段的形成，同时保持该蛋白质分子的活性生物学构象。
发明内容
本申请提供了一种液体制剂，所述液体制剂可包含重组hEPO-Fc融合蛋白、缓冲液和稳定剂，还可包含表面活性剂，或pH调节剂。所述液体制剂具有稳定性，并且在不同放置条件下，例如，光照、高温、震荡和/或冻融条件下，所述重组hEPO-Fc融合蛋白在所述制剂中形成蛋白聚体或片段的含量少，仍保持其物理稳定性、化学稳定性和/或生物学活性的制剂。所述的液体制剂具有以下一种或多种性质：(1)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的聚体增加低于10％；(2)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的片段增加低于8％；(3)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的纯度至少为90％。
一方面，本申请提供了一种液体制剂，其包含重组hEPO-Fc融合蛋白、缓冲液和稳定剂，其中，所述重组hEPO-Fc融合蛋白包含SEQ ID NO:1所示的氨基酸序列，且所述重组hEPO-Fc融合蛋白的浓度为0.1mg/ml-10mg/ml。
在某些实施方式中，所述缓冲液选自下组中的一种或多种：磷酸盐缓冲液、Tris缓冲液和组氨酸盐酸盐缓冲液(His/HCl)。
在某些实施方式中，所述缓冲液选自下组中的一种或多种：磷酸盐缓冲液和Tris缓冲液。
在某些实施方式中，所述缓冲液为磷酸盐缓冲液。
在某些实施方式中，所述缓冲液的浓度为约1mM-50mM。在某些实施方式中，所述缓冲液的浓度为约1mM-10mM。在某些实施方式中，所述缓冲液的浓度为约10mM。
在某些实施方式中，所述稳定剂包括精氨酸或其药学上可接受的盐。在某些实施方式中，所述稳定剂为精氨酸盐酸盐。
在某些实施方式中，所述稳定剂的浓度为约10mM-500mM。在某些实施方式中，所述稳定剂的浓度为约10mM-150mM。在某些实施方式中，所述稳定剂的浓度为约150mM。
在某些实施方式中，所述液体制剂包括表面活性剂。
在某些实施方式中，所述表面活性剂包括聚山梨酯。
在某些实施方式中，所述表面活性剂包括聚山梨酯20和/或聚山梨酯80。
在某些实施方式中，所述聚山梨酯20的含量为约0.0001％-0.1％(w/w)。在某些实施方式中，所述聚山梨酯20的含量为约0.001％-0.01％(w/w)。在某些实施方式中，所述聚山梨酯20的含量为约0.001％-0.05％(w/w)。在某些实施方式中，所述聚山梨酯20的含量为约0.01％-0.1％(w/w)。在某些实施方式中，所述聚山梨酯20的含量为约0.001％-0.01％(w/w)。在某些实施方式中，所述聚山梨酯20的含量为约0.01％(w/w)。
在某些实施方式中，所述稳定剂进一步包括糖类，所述糖类包括选自下组中的一种或多种：蔗糖、海藻糖和环糊精。
在某些实施方式中，所述糖类包括选自下组中的一种或多种：蔗糖和海藻糖。
在某些实施方式中，所述糖类的含量为约1％-50％。在某些实施方式中，所述糖类的含量为约2％-5％。
在某些实施方式中，所述的液体制剂进一步包括螯合剂。
在某些实施方式中，所述螯合剂包括EDTA。
在某些实施方式中，所述EDTA的浓度为约0.001mM至20mM。在某些实施方式中，所述EDTA的浓度为约5mM至10mM。
在某些实施方式中，所述液体制剂可包括pH调节剂，所述pH调节剂使得所述液体制剂的pH为约6.0-9.0。在某些实施方式中，所述的液体制剂的pH为约6.5-8.5。在某些实施方式中，所述的液体制剂的pH为约7-8。
在某些实施方式中，所述的液体制剂具有以下一种或多种性质：
(1)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的聚体增加低于10％；
(2)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的片段增加低于8％；
(3)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的纯度至少为90％。
另一方面，本申请提供了所述的液体制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗贫血。
另一方面，本申请提供了所述的液体制剂，其用于预防、缓解和/或治疗贫血。
另一方面，本申请提供了预防、缓解和/或治疗贫血的方法，其包括使用所述的液体制剂。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是不同缓冲液的液体制剂在不同放置条件下稳定性的SE-HPLC检测结果。
图2显示的是不同缓冲液的液体制剂在不同放置条件下稳定性的rCE-SDS检测结果。
图3显示的是不同缓冲液的液体制剂在不同放置条件下稳定性的nrCE-SDS检测结果。
图4显示的是不同缓冲液的液体制剂在不同放置条件下稳定性的nrCE-SDS检测结果，其中，A：低分子量片段(LMWS)含量，B：高分子量聚体(HMWS)含量，C：28min片段含量，D：24min片段含量。
图5显示的是不同缓冲液的液体制剂在震荡条件下稳定性的icIE检测结果。
图6显示的是不同缓冲液的液体制剂在光照条件下稳定性的icIE检测结果。
图7显示的是不同缓冲液的液体制剂在37C/2W下稳定性的icIE检测结果。
图8显示的是不同辅料的液体制剂在对照(A)、25C/2W(B)和冻融(C)条件下的SE-HPLC检测结果。
图9显示的是不同辅料的液体制剂在37/2W下的高分子量聚体含量。
图10显示的是同一缓冲体系添加不同辅料的液体制剂稳定性的SE-HPLC检测结果。
图11显示的是不同辅料的液体制剂在不同条件下稳定性的SE-HPLC检测结果。
图12显示的是不同辅料的液体制剂在不同条件下HMWS含量。
图13显示的是不同辅料的液体制剂在不同条件下LMWS含量
图14显示的是添加PS20或环糊精的液体制剂在不同条件下不溶性颗粒的粒径大小。
图15显示的是添加NaCl或精氨酸盐酸盐的液体制剂在不同条件下不溶性颗粒的粒径大小。
图16显示的是不同种类和浓度的表面活性剂的液体制剂的不溶性颗粒的粒径大小，其中SK：室温震荡；RT：室温静置。
图17显示的是不同浓度的磷酸盐缓冲液下SE-HPLC检测的液体制剂主峰含量。
图18显示的是不同浓度的磷酸盐缓冲液下SE-HPLC检测的液体制剂HMWS含量。
图19显示的是不同浓度的磷酸盐缓冲液下SE-HPLC检测的液体制剂LMWS含量。
图20显示的是不同浓度的Arg-HCl下SE-HPLC检测的液体制剂主峰含量。
图21显示的是不同浓度的Arg-HCl下SE-HPLC检测的液体制剂HMWS含量。
图22显示的是不同浓度的Arg-HCl下SE-HPLC检测的液体制剂LMWS含量。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“重组hEPO-Fc融合蛋白”通常是指由人促红细胞生成素(hEPO)和人IgG的Fc片段融合而成的融合蛋白。促红细胞生成素(EPO)是约30.4千道尔顿的糖蛋白激素，其可以促进红细胞祖细胞的增生并维持它们分化成成熟的红细胞。重组人EPO(rHuEPO)己广泛应用于在晚期和透析前阶段因肾病引起的慢性贫血患者的治疗中。所述IgG可以是IgG2。所述hEPO与人IgG的Fc片段可以通过肽接头间接融合。重组hEPO-Fc融合蛋白中的2个hEPO-IgG2Fc融合蛋白经二硫键在IgG2Fc区域结合，产生了连接在Fc片段N末端的EPO二聚体。重组hEPO-Fc融合蛋白可包含糖基化位点和二硫键。例如，所述重组hEPO-Fc蛋白可包含如下的氨基酸序列：
Figure PCTCN2021111804-appb-000001
Figure PCTCN2021111804-appb-000002
在本申请中，术语“稳定性”、“稳定”通常是指是指包含蛋白(例如，重组hEPO-Fc融合蛋白)的液体制剂中，蛋白(例如，重组hEPO-Fc融合蛋白)在给定的生产、制备、运输和/或贮存条件下不发生、或仅极少地发生聚集、降解或片段化。“稳定”的液体制剂在给定的生产、制备、运输和/或贮存条件下保持生物学活性。可通过例如尺寸排阻高效液相色谱(SE-HPLC)、还原毛细管电泳(rCE-SDS)法、非还原性毛细管电泳(nrCE-SDS)法、全柱成像毛细管等电聚焦电泳(icIEF)法和/或粒径(PM)检测技术测量所述制剂的聚集、降解或片段化程度等，从而评估所述蛋白(例如，重组hEPO-Fc融合蛋白)的稳定性。在本申请中，低于重组hEPO-Fc融合蛋白分子量的蛋白可以认为是片段，高于重组hEPO-Fc融合蛋白分子量的蛋白可以认为是聚体。
在本申请中，术语“糖类”通常可以包含不同量的糖(糖(saccharide))单位，例如单糖、二糖、三糖、四糖、五糖和多糖。糖类还包括他们的衍生物，例如氨基糖、糖醇、醛糖等。糖类的例子可包括葡萄糖、果糖、半乳糖、甘露糖、山梨糖、核糖、脱氧核糖、蔗糖、海藻糖、乳糖、麦芽糖、麦芽糊精、右旋糖酐(Dextran)、环糊精和/或淀粉。
在本申请中，术语“聚山梨酯”通常是指山梨醇及其脱水物的油酸酯，例如，与环氧乙烷共聚的。聚山梨酯可包括聚山梨酯20(聚环氧乙烷(20)失水山梨醇单月桂酸酯，吐温20，或PS20)，和聚山梨酯80(聚环氧乙烷(80)失水山梨醇单月桂酸酯，吐温80或PS80)。
在本申请中，术语“贫血”通常是指一种宽泛限定的状况，其特征在于血液中的血红蛋白或红细胞低于正常水平。在一些情况下，贫血可以是由红细胞的生成或存活中的原发性病症所引起的。更通常地，贫血继发于其它系统的疾病(Weatherall&amp；Provan(2000)Lancet355，1169-1175)。贫血可由红细胞生成速率下降或破坏速率上升导致，或者由因出血引起的红细胞丧失导致。贫血可由多种病症引起，所述病症包括，例如，慢性肾功能衰竭、化学疗法治疗、骨髓异常增生综合征、类风湿关节炎以及骨髓移植。例如，所述贫血可以是由无效性红细胞生成疾病引起的。
在本申请中，术语“w/w”通常是指“质量体积比”，为组分的质量与制剂的体积比。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”或“包括”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
一方面，本申请提供了一种液体制剂，其包含重组hEPO-Fc融合蛋白，所述重组hEPO-Fc融合蛋白可包含如下所示的氨基酸序列：
Figure PCTCN2021111804-appb-000003
或者所述重组hEPO-Fc融合蛋白可包含与SEQ ID NO:1所示的氨基酸序列具有至少70％(例如，至少75％、80％、85％、90％、95％、96％、97％、98％、99％或更多)序列同一性的氨基酸序列。在某些情形中，所述重组hEPO-Fc融合蛋白的浓度可以为约0.1mg/ml-10mg/ml，例如，所述重组hEPO-Fc融合蛋白的浓度可以为约0.1mg/ml-9mg/ml、约0.1mg/ml-8mg/ml、约0.1mg/ml-7mg/ml、约0.1mg/ml-6mg/ml、约0.1mg/ml-5mg/ml、约0.1mg/ml-4mg/ml、约0.5mg/ml-4mg/ml、约0.5ml/ml-3mg/ml或约0.5mg/ml-2mg/ml。例如，所述重组hEPO-Fc融合蛋白的浓度可以为约2mg/ml。所述重组hEPO-Fc融合蛋白可包含两条上述氨基酸序列组成的蛋白二聚体。
在本申请中，所述液体制剂可包含缓冲液，缓冲液可以选自下组中的一种或多种：磷酸盐缓冲液、Tris缓冲液和组氨酸盐酸盐缓冲液(His/HCl)。需要说明的是，在本申请中，如涉及到制剂中含有缓冲液或者缓冲体系，也是指制剂中含有缓冲剂，而通过缓冲剂在所述的制剂中形成缓冲体系。在某些实施方案中，评价了不同缓冲体系如：磷酸盐缓冲液、Tris缓冲液、醋酸钠缓冲液和组氨酸盐酸盐缓冲液(His/HCl)对所述重组hEPO-Fc融合蛋白稳定性的影响。在上述缓冲液中，磷酸盐缓冲液、Tris缓冲液和组氨酸盐酸盐缓冲液(His/HCl)的缓 冲效果好，醋酸钠缓冲液的效果差。例如，所述缓冲液可以选自下组中的一种或多种：磷酸盐缓冲液和Tris缓冲液。例如，所述缓冲液可以为磷酸盐缓冲液。在本申请中，所述磷酸盐缓冲液可包括磷酸钠缓冲液(可包括磷酸二氢钠、磷酸氢二钠)、磷酸钾缓冲液或以上两者的组合。又例如，所述缓冲液可以为Tris缓冲液。在某些情形中，所述缓冲液可以包含选自下组中的两种或两种以上缓冲液组成的交叉缓冲液：磷酸盐缓冲液、Tris缓冲液和组氨酸盐酸盐缓冲液(His/HCl)。例如，所述缓冲液可以是His/Tris缓冲液。所述的缓冲剂可以起到缓冲外来pH变化的作用，使溶液的pH值保持在一定的范围内。
在本申请中，所述液体制剂中的所述缓冲液由缓冲剂形成，所述缓冲液的浓度可以为约1mM-50mM。例如，所述缓冲液的浓度可以为约1mM-45mM、约1mM-40mM、约1mM-35mM、约1mM-30mM、约1mM-25mM、约1mM-20mM、约1mM-15mM、约2mM-15mM、约2mM-20mM或约5mM-20mM。例如，所述液体制剂中的所述缓冲液的浓度可以为约10mM。
在本申请中，所述液体制剂还可包括pH调节剂，所述pH调节剂可以使得所述液体制剂的pH为约6.0-9.0，例如，为约6.5-8.5、为约6.5-8.0、或为约6.5-7.5。在本申请中，所述的液体制剂的pH可以为7.0-8.0。在本申请中，所述的液体制剂的pH可以为约6.5、约7.0、约7.5、约8.0或约8.5。例如，所述的液体制剂的pH可以为约7.0。所述pH调节剂可以是有机酸或无机酸、碱、中和剂和/或缓冲剂。
在本申请中，所述液体制剂还可包含一种或多种合适的稳定剂，其可为药用赋形剂，在制造、存储和/或使用过程中保护药物活性成分(例如，所述重组hEPO-Fc融合蛋白)免于化合和/或物理降解。由于蛋白质的分子量较大，氨基酸发生降解的情况也较多，各种物理化学作用能够引发氨基酸发生去氨基化、环化、化学键断裂等变化。在某些情形中，所述稳定剂包括但不限于，合适的糖类、氨基酸、多元醇或其合适的衍生物或混合物。
可用作稳定剂的合适的氨基酸包括但不限于精氨酸、甘氨酸、赖氨酸、组氨酸、谷氨酸、天冬氨酸、异亮氨酸、亮氨酸、丙氨酸、苯丙氨酸、酪氨酸、色氨酸、甲硫氨酸、丝氨酸、脯氨酸、半胱氨酸和/或胱氨酸，或任何以上的组合以及它们药学上可接受的盐。所述氨基酸可以以氨基酸盐的形式存在，例如，氨基酸盐酸盐。例如，所合适的氨基酸可包括精氨酸，或者，所述精氨酸可以以精氨酸盐酸盐的形式添加至所述液体制剂。
在本申请中，所述氨基酸的浓度可以为约10mM-500mM。例如，所述氨基酸的浓度可以为约10mM-450mM、约10mM-400mM、约10mM-350mM、约10mM-300mM、约50mM-300mM、约50mM-250mM、约50mM-200mM或约100mM-200mM。例如，本申请所述液体制剂中的精氨酸或其药学上可接受的盐(例如，精氨酸盐酸盐)的浓度约为10-500mM。例如，本申请 所述液体制剂中的精氨酸或其药学上可接受的盐(例如，精氨酸盐酸盐)的浓度约为150mM。
在本申请中，可用作所述液体制剂的稳定剂可包括糖类。合适的糖类可包括但不限于氨基糖、糖醇、醛糖等。糖类的例子可包括葡萄糖、果糖、半乳糖、甘露糖、山梨糖、核糖、脱氧核糖、蔗糖、海藻糖、乳糖、麦芽糖、麦芽糊精、右旋糖酐(Dextran)、环糊精和淀粉，或其合适的混合物。例如，所述糖类可以包括选自下组中的一种或多种：蔗糖、海藻糖和环糊精。例如，所述糖类可包括选自下组中的一种或多种：蔗糖和海藻糖。例如，所述糖类可以包括蔗糖。
在本申请中，所述糖类的含量可以为约1％-50％，例如，约1％-50％、约1％-45％、约1％-40％、约1％-35％、约1％-30％、约1％-25％、约1％-20％、约1％-15％、约1％-10％。
在本申请中，所述液体制剂还可包括表面活性剂。所述表面活性剂可以为药用赋形剂，用于保护蛋白质(例如，所述重组hEPO-Fc融合蛋白)免受多种强化条件，例如搅拌、剪切、暴露于高温等。所述表面活性剂也可以增溶，或防止蛋白质在振荡过程中聚合，与内包材、空气、胶塞接触时吸附或者析出。所述表面活性剂可以包括但不限于聚山梨酯(吐温，或聚氧乙烯脱水山梨醇脂肪酸酯)、聚氧乙烯烷基醚(例如，Brij)、烷基苯基聚氧乙烯醚(例如，Triton-X)、聚氧乙烯-聚氧丙烯共聚物(例如，泊洛沙姆、普朗尼克)、十二烷基磺酸钠(SDS)，后上述的任意组合。例如，所述表面活性剂可包括聚山梨酯。例如，所述表面活性剂可包括聚山梨酯20和/或聚山梨酯80。
在本申请中，所述表面活性剂的含量可以为约0.0001％-0.1％(w/w)，例如，所述表面活性剂的含量可以为约0.0005％-0.1％(w/w)、约0.001％-0.1％(w/w)、约0.001％-0.09％(w/w)、约0.001％-0.08％(w/w)、约0.001％-0.07％(w/w)、约0.001％-0.06％(w/w)、约0.001％-0.05％(w/w)、约0.002％-0.04％(w/w)、约0.003％-0.03％(w/w)、约0.004％-0.025％(w/w)、约0.001％-0.045％(w/w)、约0.001％-0.04％(w/w)、约0.001％-0.035％(w/w)、约0.001％-0.03％(w/w)。例如，所述聚山梨酯20的含量可以为0.001％-0.05％(w/w)。
例如，所述液体制剂中的表面活性剂可包括聚山梨酯20和/或聚山梨酯80，且浓度为约0.001％至约0.1％(w/w)。
在本申请中，所述的液体制剂还可进一步包括螯合剂。螯合剂通常指的是有许多带负电荷的配体和/或多电子配体的多官能分子，所述配体可以以不同的亲和力多价螯合金属离子。合适的多电子官能团包括羧基、羟基和氨基。螯合剂的实例包括氨基多羧酸例如乙二胺四乙酸(EDTA)、二亚乙基三胺五乙酸(DTPA)、次氮基三乙酸(NTA)、N-2-乙酰氨基-2-亚氨基二乙酸(ADA)、二(氨乙基)乙二醇醚-N,N,N’,N’-四乙酸(EGTA)、反式-环己二胺四乙酸 (DCTA)、谷氨酸和/或天冬氨酸，羟基氨基羧酸例如N-羟乙基亚氨基二乙酸(HIMDA)、N,N-二-羟乙基甘氨酸(N-二(羟乙基)甘氨酸)和/或N-(三羟甲基甲基)甘氨酸(N-三(羟甲基)甲基甘氨酸)，以及N-取代的甘氨酸例如N-甘氨酰甘氨酸。其它的候选螯合剂可包括2-(2-氨基-2-氧代乙基)氨基乙磺酸(BES)和去铁胺(DEF)。
在本申请中，所述螯合剂可包括EDTA。例如，所述EDTA的浓度可以为约0.001mM至20mM，例如，约0.001mM至18mM、约0.001mM至16mM、约0.001mM至14mM、约0.001mM至12mM、约0.001mM至10mM、约1mM至14mM、约1mM至12mM，或约1mM至10mM。
在本申请中，所述液体制剂可包括以下物质：所述重组hEPO-Fc融合蛋白、所述缓冲液和所述稳定剂。例如，所述液体制剂可包括以下物质：浓度为约0.1mg/ml-10mg/ml所述重组hEPO-Fc融合蛋白、约1mM-50mM的磷酸盐缓冲液(PB)和浓度为约10mM-500mM的精氨酸(或精氨酸盐酸盐)，还可包括约0.0001％-0.1％(w/w)的聚山梨酯20或聚山梨酯80，和/或约0.001mM至20mM的EDTA，且pH可以为约6.0-9.0。
例如，所述液体制剂可包括以下物质：浓度为约1-5mg/ml所述重组hEPO-Fc融合蛋白、约1-20mM的磷酸盐缓冲液(PB)、浓度为约50mM-300mM的精氨酸(或精氨酸盐酸盐)和约0.001％-0.02％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-8.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约10mM的磷酸盐缓冲液(PB)和浓度为约150mM的精氨酸(或精氨酸盐酸盐)，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约10mM的磷酸盐缓冲液(PB)、浓度为约150mM的精氨酸(或精氨酸盐酸盐)和约0.005％-0.01％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约1-50mM的磷酸盐缓冲液(PB)、浓度为约10-500mM的精氨酸(或精氨酸盐酸盐)和约0.001％-0.1％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约1mM的磷酸盐缓冲液(PB)、浓度为约10-500mM的精氨酸(或精氨酸盐酸盐)和约0.001％-0.1％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约1-10mM的磷酸盐缓冲液(PB)、浓度为约10-500mM的精氨酸(或精氨酸盐酸盐)和约 0.001％-0.1％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、10mM的磷酸盐缓冲液(PB)、浓度为约150mM的精氨酸(或精氨酸盐酸盐)和约0.01％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约1-2mg/ml所述重组hEPO-Fc融合蛋白、约1-10mM的磷酸盐缓冲液(PB)、浓度为约50-150mM的精氨酸(或精氨酸盐酸盐)和约0.01％-0.1％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6.5-7.5。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、1-10mM的磷酸盐缓冲液(PB)、浓度为约50-150mM的精氨酸(或精氨酸盐酸盐)和约0.01％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约7。
又例如，所述液体制剂可包括以下物质：浓度为约2mg/ml所述重组hEPO-Fc融合蛋白、约10mM的Tris、浓度为约150mM的精氨酸(或精氨酸盐酸盐)和约0.005％-0.01％(w/w)聚山梨酯20或聚山梨酯80，且可将pH调节为约6-8。
总之，本申请所提供的液体制剂中，通过选择适当的缓冲体系、稳定剂和/或添加表面活性剂，可以有效抑制由于冻融循环、长期存储和温度、光照变化过程中酸峰、聚体、片段和不溶性微粒的增多，活性成分能够长期稳定保存。
本申请提供了一种液体制剂，其具有稳定性，并且放置在所述制剂中形成蛋白聚体或片段的含量少。一般来说，稳定的制剂是储存一定时间后保持其物理稳定性、化学稳定性和/或生物学活性的制剂。在本申请中，所述液体制剂的稳定性可以通过其中的蛋白质(例如重组hEPO-Fc融合蛋白)的含量、蛋白质片段的含量和/或蛋白质聚体的含量来评价。
在本申请中，可通过例如尺寸排阻高效液相色谱(SE-HPLC)、还原毛细管电泳(rCE-SDS)法、非还原性毛细管电泳(nrCE-SDS)法、全柱成像毛细管等电聚焦电泳(icIEF)法和/或粒径(PM)检测技术测量所述制剂中的目的蛋白质、片段和/或聚体含量，或不溶物的粒径，从而评估所述蛋白(例如，重组hEPO-Fc融合蛋白)的稳定性。
在申请中，所述的液体制剂具有以下一种或多种性质：
(1)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的聚体增加低于10％，例如，低于9％、低于8％、低于7％、低于6％、低于5％、低于4％、低于3％、低于2％、低于1％或更低；
(2)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的片段增加低于8％，例如，低于7％、低于6％、低于5％、低于4％、低于3％、低于2％、低于1％或更低；
(3)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的纯度至少为90％，例如，至少为91％、至少为92％、至少为93％、至少为94％、至少为95％、至少为96％、至少为97％、至少为98％、至少为99％或更高；
(4)在-70℃-5℃循环冷冻溶解5次条件下，所述重组hEPO-Fc融合蛋白的纯度至少为95％，例如，至少为96％、至少为97％、至少为98％、至少为99％或更高。
另一方面，本申请提供了所述的液体制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗贫血。
另一方面，本申请提供了所述的液体制剂，其用于预防、缓解和/或治疗贫血。
另一方面，本申请提供了预防、缓解和/或治疗贫血的方法，其包括使用所述的液体制剂。
在本申请中，所述液体制剂可以用于治疗地中海贫血综合征，还可用于治疗无效性红细胞生成病症。此类病症包括铁粒幼细胞性贫血(遗传性或获得性)；红细胞生成异常性贫血(I型和II型)；镰状细胞性贫血；遗传性球形红细胞增多症；丙酮酸激酶缺乏症；巨幼红细胞性贫血，可能由状况：诸如叶酸缺乏症(由于先天性疾病、摄入减少或需求增加)，钴胺素缺乏症(由于先天性疾病、恶性贫血、吸收受损、胰腺功能不全或摄入减少)，某些药物，或不明病因(先天性红细胞生成异常性贫血、难治性巨幼红细胞性贫血或红白血病)引起；骨髓病性贫血，包括骨髓纤维化(髓样化生)和骨髓痨；先天性红细胞生成性卟啉病；和/或铅中毒。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
放置条件
将按各实施例中配置好的液体制剂用0.22μm过滤装置(Millipore TM，SLGV004SL)过滤除菌，并无菌罐装至经灭菌的玻璃瓶中(肖特新康药品包装有限公司，中硼276)进行密封。将装有液体制剂的玻璃瓶放置在以下各条件下贮存后，使用上述方法进行检测。
对照条件：5℃，静置14天。震荡条件：200rpm水平方向离心14天。冻融条件：-70℃至5℃，缓慢循环5次。光照条件：4500Lux 14天，UV照射5天。25C/1W条件：25℃下静置1周。25C/2W条件：25℃下静置2周25C/1M条件：25℃下静置1个月。25C/2M条件：25℃下静置2个月。25C/3M条件：25℃下静置3个月。37C/1W条件：37℃下静置1周。37C/2W条件：37℃下静置2周。37C/1M条件：37℃下静置1个月。37C/2M条件：37℃下静置2个月。37C/3M条件：37℃下静置3个月。
检测方法
表观检测：将不同配方的样品在不同条件下放置后，裸眼观察澄清度。
尺寸排阻高效液相色谱(SE-HPLC)检测方法：Agilent高效液相色谱仪1260、紫外/可见光检测器，色谱柱TSKgel G3000SW XL(7.8x 300mm，5μm，TOSOH)，流动相40mM PB+0.3M Na 2SO4，pH6.5，流速0.6mL/min，上样体积20μg，样品室温度2-8℃，检测时间35min，吸收波长280nm，记录色谱图，积分后按面积归一化法计算LP-001的主峰、高分子量组分、低分子量组分的相对含量。
还原毛细管电泳(rCE-SDS)检测方法：
LP-001蛋白换液至10mM磷酸盐缓冲液(pH7.0)，并调整蛋白质浓度至5mg/mL。20μL 5mg/mL LP-001蛋白与50μl含1％SDS样品缓冲液(100mM Tirs-HCl，pH9.0)混匀后，加入5μL还原剂(β-巯基乙醇)和25μL超纯水，70℃孵育10mins后冷却，转移至进样小瓶中，置于Agilent公司G7100A仪器的15℃进样盘中，使用无涂层毛细管(内径50μm，有效分离长度为8.5cm)，检测波长220nm的吸光度值。
通过对电泳谱图积分，按面积归一法计算，根据校正峰面积百分比确定LP-001的主峰、高分子量组分、低分子量组分的相对含量。
非还原性毛细管电泳(nrCE-SDS)检测方法：
LP-001蛋白换液至10mM磷酸盐缓冲液(pH7.0)，并调整蛋白质浓度至5mg/mL。20μL 5mg/mL LP-001蛋白与50μL含1％样品缓冲液(100mM Tirs-HCl，pH9.0)混匀后，加入5μL烷基化试剂(1M碘乙酸)和25μL超纯水，70℃孵育10mins后冷却，转移至进样小瓶中，置于Agilent公司G7100A仪器的15℃进样盘中，使用无涂层毛细管(内径50μm，有效分离长度为8.5cm)，检测波长220nm的吸光度值。
通过对电泳谱图积分，按面积归一法计算，根据校正峰面积百分比确定LP-001的主峰、高分子量组分、低分子量组分的相对含量。
全柱成像毛细管等电聚焦电泳(icIEF)检测方法：
LP-001蛋白与终浓度为0.35％的甲基纤维素、4％3-10两性电解质、0.5％pI Marker(4.22,9.46)和4M尿素混合，使用Protein Simple公司的Maurice C.和氟碳涂层毛细管，分别在1500V聚焦1min和3000V聚焦10mins，波长280nm下对LP-001蛋白的电荷变异体进行检测。按照按面积归一法计算，报告主峰组分、酸性峰组分和碱性峰组分的相对含量
粒径(PM)检测方法：
取4支供试品，室温静置1小时，用超纯水将容器外壁洗净，轻轻上下翻转样品20次，使溶液混合均匀，静置≥2分钟脱气泡，小心开启容器，使用不溶性微粒检测仪(Particle Sizing  System；型号：A2000usp)对供试品抽取0.8mL进行检测，共测定4次，弃第1次测定数据，取后3次测定数据的平均值作为测定结果。
在本申请的实施例中，所述精氨酸均以精氨酸盐酸盐的形式添加至所述液体制剂。
实施例1 不同pH和缓冲液对液体制剂稳定性的影响
根据以下表1配方配置液体制剂，调节pH。
表1 制剂配方
Figure PCTCN2021111804-appb-000004
将各组制剂分别于对照、冻融、震荡、光照和37C/14D条件下放置一段时间后，进行检测。
(1)SE-HPLC结果显示，醋酸钠pH为5的缓冲液中蛋白质不稳定，PB缓冲液和His/HCl缓冲液中蛋白质稳定。图1显示的是冻融条件下各组的SE-HPLC检测结果，除醋酸钠配方(组5)外，其它配方在-70℃至5℃冻融循环5次条件下，与对照(5C/14天)相比，主峰百分比无显著变化；组5主峰百分比明显降低，说明液体制剂中的蛋白质分子量发生了变化，例如生成了聚体或片段。
还检测了高分子量组分(HMWS)和低分子量组分(LMWS)的百分比，组1在各筛选条件下的HMWS或LMWS生成情况如下表2所示。SE-HPLC检测结果可知，PB缓冲液pH＝7环境下，液体制剂均稳定。
表2 PB7组在各筛选条件下的SE-HPLC结果
条件 主峰 高分子量组分HMWS(％) 低分子量组分LMWS(％)
对照 99.5 0.5 低于检测限
冻融 99.5 0.5 低于检测限
震荡 95.4 1.2 3.4
光照 80.4 19.6 低于检测限
37C/14D 83.9 9.5 6.6
(2)rCE-SDS结果与SE-HPLC结果一致，组5在震荡、光照和37℃14天放置下不稳定，而pH＝6或7时的PB和His/HCl缓冲液稳定。如图2所示。在震荡、光照和37C14D筛选条件下PB7组显著优于其他组，在冻融筛选条件下各组间无显著差别(图2)。
也检测了HMWS和LMWS的百分比，组1(PB缓冲液，pH＝7)在各筛选条件下的rCE-SDS检测结果如下表3所示，说明蛋白质分子在液体制剂中稳定，生成聚体或片段少。
表3 组1在各筛选条件下的rCE-SDS结果
条件 主峰 高分子量聚体HMWS(％) 低分子量片段LMWS(％)
对照 100.0 低于检测限 低于检测限
冻融 99.8 0.2 低于检测限
震荡 98.5 低于检测限 1.5
光照 99.4 低于检测限 0.6
37C/14D 98.5 低于检测限 1.5
(3)nrCE-SDS结果如图3和图4所示。在各放置条件下，观察到偏酸性的组3和组5中，主峰下降显著(图3)。图4显示了在光照和37C/14D条件下各组的LMWS和HMWS的变化；从图4A可知，pH＝5时，液体制剂不稳定，易形成低分子量片段。图4C和图4D显示，其中LMWS主要集中在28min和24min的片段峰(主峰为32min)；根据出峰时间，推测24min对应的可能是单链峰。
组1(PB缓冲液，pH＝7)在各筛选条件下的nrCE-SDS检测结果如下表4所示，说明蛋白质分子在液体制剂中稳定，生成聚体或片段少。
表4 组1在各筛选条件下的nrCE-SDS结果
条件 主峰 高分子量聚体HMWS(％) 低分子量片段LMWS(％)
对照 98.9 低于检测限 1.1
冻融 98.5 低于检测限 1.5
震荡 98.3 低于检测限 1.7
光照 98.0 2.0 0.0
37C/14D 95.7 低于检测限 4.3
(4)icIEF结果如图5-7所示。在震荡(图5)、光照(图6)和37C/2W(图7)条件下，组1、2和4比组3和5稳定。说明pH＝5时醋酸钠缓冲液的液体制剂不稳定，pH＝6或7时PB缓冲液和His/HCl缓冲液的液体制剂稳定。
实施例2 不同辅料对液体制剂稳定性的影响
根据以下表5配方配置液体制剂，并调节pH。
表5 制剂配方
Figure PCTCN2021111804-appb-000005
Figure PCTCN2021111804-appb-000006
将各组制剂分别于不同放置条件下放置一段时间后，进行检测。其中，PB缓冲体系中pH为6.0、7.0和8.0的组分别称为PB6、PB7和PB8，Tris缓冲体系中pH为8.0的组称为Tris8。
(1)SE-HPLC检测结果如图8-9所示。
图8A-8C显示的分别是各组在对照(A)、25C/2W(B)、冻融(C)条件下SE-HPLC检测的主峰百分比。结果显示，在25℃放置2周后，PB缓冲体系和Tris缓冲体系下，pH＝7和pH-8时液体制剂中的蛋白稳定性良好，在pH＝6的PB缓冲体系中，添加了甘氨酸和山梨糖醇的液体制剂中蛋白主峰降低至80％以下(图8B)。在冻融循环5次后，各缓冲体系不同pH下，添加了甘氨酸的液体制剂的蛋白主峰降低(图8C)。
同时，使用SE-HPLC检测高分子量聚体的含量，结果显示在PB缓冲体系pH为6时，37℃下放置2周后，加入精氨酸盐酸盐的液体制剂中的蛋白质生成的聚体少，说明精氨酸盐酸盐能降低蛋白质聚体的形成(图9)。
图10显示的是在PB7体系中，加入不同稳定剂，分别置于不同的条件下，SE-HPLC的检测结果。结果显示精氨酸盐酸盐、山梨糖醇、蔗糖和海藻糖的组的主峰百分比高于不加稳定剂的对照组，加入甘氨酸的组的主峰百分比低于对照组，说明精氨酸盐酸盐、山梨糖醇、蔗糖和海藻糖对蛋白质的稳定效果好，且并不是任意一种稳定剂加入后都能使蛋白质稳定，甘氨酸反而不利于稳定。
实施例3 不同辅料对液体制剂稳定性的影响
根据以下表6配方配置液体制剂，调节pH。
表6 制剂配方
Figure PCTCN2021111804-appb-000007
Figure PCTCN2021111804-appb-000008
将各组制剂分别于不同放置条件下放置一段时间后，进行检测。其中，PB缓冲体系中pH为6.0、7.0和8.0的组分别称为PB6、PB7和PB8，Tris缓冲体系中pH为8.0的组称为Tris8。
(1)表观检测有无可见颗粒的形成情况，结果如下表7所示。
表7 表观检测结果
Figure PCTCN2021111804-appb-000009
表观检测的结果显示，对比组2和组3，或组3和组4，可以发现，加入PS20可以避免可见颗粒的形成，而加入环糊精和右旋糖酐不利于液体制剂的稳定。
(2)SEC-HPLC结果如图11-13所示。SEC-HPLC检测的结果显示，组1、组2、组3、组5、组6和组9在25℃、37℃或震荡下放置较长时间均稳定，主峰含量基本不变(图11)，说明精氨酸盐酸盐和环糊精、EDTA或PS-20的组合稳定。且精氨酸盐酸盐与PS20的组中，高分子量聚体和低分子量片段都较少，说明蛋白质稳定，不易聚集或降解(图12和图13)。而加入右旋糖酐的液体制剂(组4和组8)在各条件下不稳定，生成的蛋白质聚体或片段较多(图12和图13)。且包含Tris缓冲液和精氨酸盐酸盐的液体制剂在各条件下主峰含量变化也小，不易生成片段(图12和图13)。结果还显示，包含氯化钠(NaCl)或精氨酸盐酸盐的液体制剂也稳定(组1)。
(3)PM结果如图14-15所示。图14显示的是组3和组5，比较加入PS20的组3和组5的PM结果，加入环糊精的组5，在各条件下放置一定时间后，组5产生的分子粒径较大，说明蛋白质生成了聚体，而包含精氨酸盐酸盐和PS-20的组3则稳定，不易生成聚体(图14)。图15显示的是加入氯化钠的组1和精氨酸盐酸盐的组3在不同放置条件下的PM结果。比较加入氯化钠的组1和加入精氨酸盐酸盐的组3，在各条件下放置一定时间后，组1生成的分子粒径较大，说明蛋白质生成了聚体(图15)，而组3的大粒径的分子较少。综合来看，包含精氨酸盐酸盐和PS20的液体制剂较稳定。
实施例4 不同浓度的辅料对液体制剂稳定性的影响
根据以下表8配方配置液体制剂，调节pH。
表8 制剂配方
Figure PCTCN2021111804-appb-000010
将各组制剂分别于不同放置条件下放置一段时间后，进行检测。其中，PB缓冲体系中pH为6.0、7.0和8.0的组分别称为PB6、PB7和PB8，Tris缓冲体系中pH为8.0的组称为Tris8。
(1)SE-HPLC检测蛋白质含量和生成的聚体或片段的含量，结果如下表9。所有组在 25℃下放置2周，液体制剂均能稳定存在，至少95％的蛋白质分子不会降解为片段或形成聚体；在37℃下放置2周，液体制剂也能稳定存在，至少90％的蛋白质分子不会降解为片段或形成聚体，片段生成量不超过4.0％，聚体生成量不超过7％。
表9 SE-HPLC检测结果
Figure PCTCN2021111804-appb-000011
Figure PCTCN2021111804-appb-000012
(2)nrCE-SDS检测蛋白质含量和生成的聚体或片段的含量，结果如下表10。所有组在25℃或37℃下放置2周，液体制剂均能稳定存在，至少95％的蛋白质分子不会降解为片段或形成聚体。即使在37℃下存放2周，片段生成量不超过5％。
表10 nrCE-SDS检测结果
Figure PCTCN2021111804-appb-000013
Figure PCTCN2021111804-appb-000014
其中，“ND”表示在检测条件下检测不到结果。
(3)rCE-SDS检测蛋白质含量和生成的聚体或片段的含量，结果如下表11。所有组在25℃或37℃下放置2周，液体制剂均能稳定存在，至少95％的蛋白质分子不会降解为片段或形成聚体。即使在37℃下存放2周，片段生成量不超过4％。
表11 rCE-SDS检测结果
Figure PCTCN2021111804-appb-000015
Figure PCTCN2021111804-appb-000016
其中，“ND”表示在检测条件下检测不到结果。
(4)icIEF检测蛋白质酸性峰、主峰和碱性峰的含量，结果如下表12。所有组在25℃或37℃下放置2周，与对照条件无显著差异，说明各组液体制剂聚能稳定存在。
表12 rCE-SDS检测结果
Figure PCTCN2021111804-appb-000017
Figure PCTCN2021111804-appb-000018
(5)PM检测液体制剂中蛋白的粒径，结果如下表13所示。所有组在25℃或37℃下放置2周，与对照条件无显著差异，说明各组液体制剂聚能稳定存在。
表13 粒径结果
Figure PCTCN2021111804-appb-000019
Figure PCTCN2021111804-appb-000020
实施例5 表面活性剂种类及浓度对液体制剂稳定性的影响
将2mg/mL的LP-001换液至10mM PB+150mM精氨酸盐酸盐(Arg-HCl)中，pH＝7.0，然后添加下表14中相应的表面活性剂。
表14 表面活性剂种类与含量信息表
Figure PCTCN2021111804-appb-000021
Figure PCTCN2021111804-appb-000022
以1mL/支的量进行分装，每组一支200rpm摇晃条件下放置7天，一支静置7天作为对照。然后进行不溶性微粒检测。结果如图16所示，PS20和PS80在0.001％至0.1％浓度范围内，大部分颗粒的直径均小于10μm，直径大于25μm的颗粒数约为0。本实施例的制剂配方均符合法规要求，即直径大于等于10μm的颗粒数小于6000粒/瓶；直径大于等于25μm的颗粒数小于600粒/瓶。
实施例6 不同浓度的磷酸盐缓冲液和辅料(精氨酸盐酸盐)对液体制剂稳定性的影响
将2mg/mL的LP-001换液至下表各配方中，对磷酸盐缓冲液(1～50mM)和精氨酸盐酸盐(Arg-HCl，10～500mM)的浓度进行研究。
表15 制剂配方
Figure PCTCN2021111804-appb-000023
以1mL/支的量进行分装，每组一支37℃条件下放置14天(37C组)，一支2-8℃条件下放置14天作为对照(5C组)。使用SE-HPLC检测纯度。
不同摩尔浓度的磷酸盐缓冲液下的结果如表16和图17至图19所示，从LP-001的主峰相对含量来看，在37℃下放置14天，LP-001的主峰纯度均在90％以上，且1mM PB的缓冲液下的稳定性优于50mM PB和10mM PB。
表16 磷酸盐缓冲液摩尔浓度筛选结果
Figure PCTCN2021111804-appb-000024
不同摩尔浓度的Arg-HCl下的结果如表17和图20至图22所示，从LP-001的主峰相对含量来看，在37℃下放置14天，随着精氨酸盐酸盐浓度的上升，主峰含量逐渐升高，在10mM至500mM之间，且主峰含量均在90％以上。
表17 精氨酸盐酸盐摩尔浓度筛选结果
Figure PCTCN2021111804-appb-000025

Claims (34)

  1. 一种液体制剂，其包含重组hEPO-Fc融合蛋白、缓冲液和稳定剂，其中，所述重组hEPO-Fc融合蛋白包含SEQ ID NO:1所示的氨基酸序列，且所述重组hEPO-Fc融合蛋白的浓度为约0.1mg/ml-10mg/ml。
  2. 根据权利要求1所述的液体制剂，其中所述缓冲液选自下组中的一种或多种：磷酸盐缓冲液(PB)、Tris缓冲液、组氨酸盐酸盐缓冲液(His/HCl)。
  3. 根据权利要求1-2中任一项所述的液体制剂，其中所述缓冲液为磷酸盐缓冲液(PB)。
  4. 根据权利要求1-3中任一项所述的液体制剂，其中所述缓冲液的浓度为约1mM-50mM。
  5. 根据权利要求1-4中任一项所述的液体制剂，其中所述缓冲液的浓度为约1mM-10mM。
  6. 根据权利要求1-5中任一项所述的液体制剂，其中所述缓冲液的浓度为约10mM。
  7. 根据权利要求1-6中任一项所述的液体制剂，其中所述稳定剂包括精氨酸或其药学上可接受的盐。
  8. 根据权利要求1-7中任一项所述的液体制剂，其中所述稳定剂为精氨酸盐酸盐(Arg-HCl)。
  9. 根据权利要求1-8中任一项所述的液体制剂，其中所述稳定剂的浓度为约10mM-500mM。
  10. 根据权利要求1-9中任一项所述的液体制剂，其中所述稳定剂的浓度为约10mM-150mM。
  11. 根据权利要求1-10中任一项所述的液体制剂，其中所述稳定剂的浓度为约150mM。
  12. 根据权利要求1-7中任一项所述的液体制剂，其可包括表面活性剂。
  13. 根据权利要求12所述的液体制剂，其中所述表面活性剂包括聚山梨酯。
  14. 根据权利要求12或13所述的液体制剂，其中所述表面活性剂包括聚山梨酯20(PS20)和/或聚山梨酯80(PS80)。
  15. 根据权利要求12-14中任一项所述的液体制剂，其中所述表面活性剂的含量为约0.0001％-0.1％(w/w)。
  16. 根据权利要求12-15中任一项所述的液体制剂，其中所述表面活性剂的含量为约0.001％-0.1％(w/w)。
  17. 根据权利要求12-16中任一项所述的液体制剂，其中所述表面活性剂的含量为约0.001％-0.01％(w/w)。
  18. 根据权利要求12-16中任一项所述的液体制剂，其中所述表面活性剂的含量为约0.01％-0.1％(w/w)。
  19. 根据权利要求12-17中任一项所述的液体制剂，其中所述表面活性剂的含量为约0.1％ (w/w)。
  20. 根据权利要求1-19中任一项所述的液体制剂，其中所述稳定剂包括糖类，所述糖类包括选自下组中的一种或多种：蔗糖、海藻糖和环糊精。
  21. 根据权利要求20所述的液体制剂，其中所述糖类的含量为约1％-50％。
  22. 根据权利要求20-21中任一项所述的液体制剂，其中所述糖类的含量为约1％-10％。
  23. 根据权利要求20-22中任一项所述的液体制剂，其中所述糖类的含量为约2％-5％。
  24. 根据权利要求1-23中任一项所述的液体制剂，其进一步包括螯合剂。
  25. 根据权利要求24所述的液体制剂，其中所述螯合剂包括EDTA。
  26. 根据权利要求24-25中任一项所述的液体制剂，其中所述螯合剂的浓度为约0.001mM至20mM。
  27. 根据权利要求24-26中任一项所述的液体制剂，其中所述螯合剂的浓度为约5mM至10mM。
  28. 根据权利要求1-27中任一项所述的液体制剂，其还包括pH调节剂，所述pH调节剂使得所述液体制剂的pH为约6.0-9.0。
  29. 根据权利要求1-28中任一项所述的液体制剂，其还包括pH调节剂，所述pH调节剂使得所述液体制剂的pH为约7.0-8.0。
  30. 根据权利要求1-29中任一项所述的液体制剂，其具有以下一种或多种性质：
    (1)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的聚体增加低于10％；
    (2)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的片段增加低于8％；
    (3)在37℃可以贮存至少2周，且其中所述重组hEPO-Fc融合蛋白的纯度至少为90％。
  31. 权利要求1-30中任一项所述的液体制剂在制备药物中的用途，所述药物用于预防、缓解和/或治疗贫血。
  32. 权利要求1-30中任一项所述的液体制剂，其用于预防、缓解和/或治疗贫血。
  33. 预防、缓解和/或治疗贫血的方法，其包括向有需要的受试者施用权利要求1-30中任一项所述的液体制剂。
  34. 融合蛋白，其包含SEQ ID NO：1所示的氨基酸序列。
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