WO2022143999A1 - 包含可溶性gp130二聚体的制剂和使用方法 - Google Patents

包含可溶性gp130二聚体的制剂和使用方法 Download PDF

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WO2022143999A1
WO2022143999A1 PCT/CN2021/143870 CN2021143870W WO2022143999A1 WO 2022143999 A1 WO2022143999 A1 WO 2022143999A1 CN 2021143870 W CN2021143870 W CN 2021143870W WO 2022143999 A1 WO2022143999 A1 WO 2022143999A1
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aqueous formulation
fusion protein
trehalose
histidine
colitis
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PCT/CN2021/143870
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English (en)
French (fr)
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陆树云
张哲如
乔君华
朱静
王静
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天境生物科技(杭州)有限公司
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Priority to CA3203432A priority Critical patent/CA3203432A1/en
Priority to CN202180087571.1A priority patent/CN116829170A/zh
Priority to EP21914727.9A priority patent/EP4272753A1/en
Priority to AU2021413653A priority patent/AU2021413653A1/en
Priority to JP2023540018A priority patent/JP2024503299A/ja
Priority to BR112023012894A priority patent/BR112023012894A2/pt
Priority to KR1020237024358A priority patent/KR20230128033A/ko
Publication of WO2022143999A1 publication Critical patent/WO2022143999A1/zh

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    • 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
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • 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
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • 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
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention belongs to the field of biomedical research, and in particular relates to a preparation comprising gp130 dimer and its use for treating various IL-6-mediated disorders, including inflammatory diseases and cancer.
  • Glycoprotein 130 (also known as gp130, IL6ST, IL6-beta or CD130) is a transmembrane protein. It forms a subunit of the type I cytokine receptor in the IL-6 receptor family. It is important for signal transduction following cytokine involvement. Structurally, gp130 consists of five fibronectin type III domains and one immunoglobulin-like C2 type domain in its extracellular portion.
  • IL-6 receptor family all form complexes with gp130 for signal transduction.
  • IL-6 binds to the IL-6 receptor.
  • the complex of these two proteins then associates with gp130.
  • the complex consisting of the 3 proteins then homodimerizes to form a hexameric complex that can generate downstream signals.
  • IL-6 is a pleiotropic cytokine produced by hematopoietic and non-hematopoietic cells, eg, in response to infection and tissue damage.
  • IL-6 exerts its multiple biological activities through two major signal transduction pathways, a so-called classical ligand-receptor pathway through which membrane-bound IL-6R is found primarily on hepatocytes and certain leukocytes, and a It is a trans-signaling pathway (trans-signaling pathway) through proteolytic cleavage derived from membrane-bound IL-6R or circulating sIL-6R (soluble IL-6R, or soluble IL-6R) derived from alternative splicing.
  • trans-signaling pathway trans-signaling pathway
  • IL-6 binds directly to membrane-bound IL-6R on the surface of a limited range of cell types.
  • the IL-6/IL-6R complex associates with pre-formed dimers of the signaling gp130 receptor protein, causing spatial changes in gp130 homodimers and thereby initiating intracellular signaling cascades.
  • Classical signaling is responsible for acute inflammatory defense mechanisms and key physiological IL-6 functions, such as intestinal epithelial cell growth and regeneration signals.
  • the extracellular domains of IL-6R and gp130 can be generated by translation of alternatively spliced mRNA without the membrane-anchoring domain, resulting in sIL-6R and gp130 variants.
  • the activity of the IL-6/sIL-6R complex is generally governed by the presence of high levels of soluble sgp130 (soluble gp130) in the circulation, which effectively competes with membrane-bound gp130.
  • soluble gp130 soluble gp130
  • the gp130 dimers of the present invention have higher binding affinity than native sgp130 and thus have a stronger ability to inhibit IL-6 signaling.
  • the formulations of the present invention allow the gp130 dimer to be more stable in production, transport and use.
  • the present invention describes a preparation comprising a gp130 dimer (or "a fusion protein comprising gp130", or “fusion protein” for short), and a preparation for the treatment of various IL- 6 Use in mediated disorders, including inflammatory diseases and cancer.
  • the formulation contains a histidine buffer system, is highly stable at around pH 7.6, and can be safely administered to humans at various doses.
  • the present specification describes an aqueous formulation (also referred to as a liquid formulation) and a lyophilized formulation comprising a fusion protein, 20-30 mM histidine, 220-280 mM trehalose and 0.01(w/v)%-0.03(w/v)% polysorbate 80, and its pH is 7.0-8.2
  • the fusion protein comprises two monomers whose amino acid sequence is SEQ ID NO: 1, and consists of connected by multiple disulfide bonds. In some embodiments, it comprises at least 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, or at least 30 mg/mL of the fusion protein.
  • the pH of the aqueous formulation is 7.4-7.8. In some embodiments, the pH of the aqueous formulation is 7.6.
  • the aqueous formulation comprises 24-26 mM histidine. In some embodiments, the aqueous formulation comprises 25 mM histidine.
  • the aqueous formulation comprises 240-260 mM trehalose. In some embodiments, the aqueous formulation comprises 250 mM trehalose.
  • the aqueous formulation comprises 0.015 (w/v) %-0.025 (w/v) % polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02 (w/v) % polysorbate 80.
  • the aqueous formulation further includes a tonicity agent (or referred to as an osmotic pressure regulator or stabilizer), a surfactant, an antioxidant, a preservative, or a mixture thereof.
  • a tonicity agent or referred to as an osmotic pressure regulator or stabilizer
  • surfactant an antioxidant, a preservative, or a mixture thereof.
  • each fusion protein molecule comprises no more than 6 galactose-alpha-1,3-galactose. In some embodiments, each fusion protein molecule comprises no more than 3, 2, or 1 galactose-alpha-1,3-galactose.
  • the fusion protein comprises glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52-65% of the glycans comprise one or more sialic acid residues.
  • the aqueous formulation comprises at least 25mg/mL of the fusion protein, 24-26mM histidine, 240-260mM trehalose and 0.015(w/v)%-0.025(w/v) )% polysorbate 80 and its pH is 7.6-7.8.
  • the aqueous formulation comprises 30 mg/mL of the fusion protein, 25 mM histidine, 250 mM trehalose and 0.02 (w/v) % polysorbate 80, and its pH is 7.6.
  • the aqueous formulation contains no more than 10 mM (or no more than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM) of amino acid salts other than histidine salts, or, preferably, , does not contain any other amino acid salts at all.
  • the aqueous formulation contains no more than 10 mM (or no more than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM) of sugars other than trehalose, or, preferably, completely Does not contain any other sugar.
  • the aqueous formulations are used to treat inflammatory diseases or IL-6 mediated disorders in humans.
  • the inflammatory disease or IL-6 mediated disorder is inflammatory bowel disease, preferably wherein the treatment induces remission of inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease or ulcerative colitis, preferably wherein the treatment maintains remission of the inflammatory bowel disease.
  • the inflammatory disease or IL-6 mediated disorder is rheumatoid arthritis, psoriasis, uveitis, or atherosclerosis.
  • the inflammatory disease or IL-6 mediated disorder is colitis unrelated to inflammatory bowel disease, preferably wherein the colitis is radiation colitis, diverticulitis, ischemic Colitis, infectious colitis, celiac disease, autoimmune colitis, or colitis caused by allergies affecting the colon.
  • This specification also captures a dry formulation that can be obtained by lyophilizing any of the aqueous formulations described herein, or adding water to produce any of the aqueous formulations described herein.
  • protein and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other linkages such as esters, ethers, and the like.
  • a protein or peptide must contain at least two amino acids and there is no restriction on the maximum number of amino acids that can constitute the sequence of the protein or peptide.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, amino acid analogs and peptidomimetics.
  • One-letter and three-letter abbreviations for naturally occurring amino acids are listed below.
  • composition is intended to mean a combination of an active agent and another inert (eg, a detectable agent or label) or active compound or composition (eg, an adjuvant).
  • “Pharmaceutical composition” is intended to include the combination of an active agent with an inert or active carrier, resulting in a composition suitable for in vitro, in vivo or ex vivo diagnostic or therapeutic use.
  • Aqueous formulation refers to a liquid formulation using water as a solvent.
  • an aqueous formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
  • buffer refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation.
  • Suitable buffers are well known in the art and can be found in the literature.
  • Pharmaceutically acceptable buffers include, but are not limited to, tris buffers, arginine buffers, histidine buffers, citrate buffers, succinate buffers, and phosphate buffers. Regardless of the buffer used, pH can be adjusted with acids or bases known in the art, such as succinic, hydrochloric, acetic, phosphoric, sulfuric and citric, succinate, citrate, tris base, histidine acid, histidine HCl, sodium hydroxide and potassium hydroxide.
  • Suitable buffers include, without limitation, histidine buffer, 2-morpholinoethanesulfonic acid (MES), cacodylate, phosphate, acetate, succinate, and citrate.
  • concentration of the buffer may be between about 4 mM and about 60 mM, or alternatively about 4 mM to about 40 mM, or alternatively about 5 mM to about 25 mM.
  • treating refers to reversing a disease or disorder or one or more symptoms thereof, alleviating a disease or disorder or one or more symptoms thereof, as described herein, Delaying the onset of or inhibiting the progression of a disease or disorder or one or more symptoms thereof.
  • treatment can be administered after one or more symptoms have developed.
  • treatment can be administered in the absence of symptoms.
  • treatment can be administered to susceptible individuals prior to the onset of symptoms (eg, taking into account a history of symptoms and/or taking into account genetic or other predisposing factors). Treatment can also be continued after symptoms have resolved, such as to prevent or delay relapse.
  • IM001 is a dimer comprising two single-chain gp130-Fc fusion proteins. It can be used to treat a variety of IL-6-mediated conditions, including inflammatory diseases and cancer. Similar to other protein drugs, the solubility, stability and activity of IM001 are affected by its environment. Therefore, it is not easy to develop a suitable formulation, including a suitable buffer system.
  • the inventors prepared 12 formulations of pH/buffer system (Table 2), 9 formulations of excipients and surfactant aqueous formulation screening formulations (Table 7), and investigated the 2-week stability at 30°C, through DSC, DLS, appearance, protein concentration , pH, SEC-HPLC, SDS (reducing/non-reducing) methods to compare them. It was found that the pH ⁇ 6.5, the foreign matter was obviously produced, and the protein was also very unstable, and it was more stable in the buffer system of pH 8.0 (alkaline). In addition, for the same pH 8.0 buffer system, the stability of histidine buffer system is stronger than that of glycine and Tris buffer system. Interestingly, the pH of the IM001 protein will drift after adding the protein to the buffer system. In the pH 8.0 histidine buffer system, the pH drift after adding the protein is 7.6.
  • the inventors of the present application prepared 5 lyophilized formulation screening formulations (Table 14), and through the stability investigation at 25°C and 40°C, the experimental results showed that the same high concentration of 30 mg/mL, The stability of the freeze-dried product was significantly improved compared with the solution formulation.
  • the lyophilized formulation (30mg/mL 25mM His, 250mM Trehalose, 0.02% PS80, pH 7.6) showed good results at different temperatures and in various tests, especially at high temperature 40°C for 2 months. There was no change, a result that was very unexpected, because in general, the same formulation did not necessarily have an advantage in various tests. Therefore, they are all formulated as lyophilized preparations of IM001 protein.
  • the sugar (trehalose) used in this lyophilized formulation is also different from the preferred version (sucrose) in the formulation of the aqueous formulation.
  • the present application provides an aqueous formulation and lyophilized formulation suitable for IM001 comprising a fusion protein, histidine salt, trehalose and polysorbate.
  • the aqueous formulation can be lyophilized to form a lyophilized formulation.
  • the lyophilized formulation after the addition of suitable water, can produce the aqueous formulation.
  • Such aqueous formulations can also be injected into a patient for the treatment of a disease for which they are compliant.
  • the fusion protein (IM001) here comprises two monomers whose amino acid sequence is SEQ ID NO: 1, connected by multiple disulfide bonds.
  • each fusion protein molecule comprises no more than 6 galactose-alpha-1,3-galactose.
  • each fusion protein molecule comprises no more than 3, 2, or 1 galactose-alpha-1,3-galactose.
  • the fusion protein comprises glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues.
  • the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52-65% of the glycans comprise one or more sialic acid residues.
  • the aqueous formulation comprises at least 10 mg/mL, 15 mg/mL, 20 mg/mL, 25 mg/mL, or at least 30 mg/mL of the fusion protein. In some embodiments, the aqueous formulation comprises 10-60 mg/mL, 15-45 mg/mL, 20-40 mg/mL, 25-35 mg/mL, or 30 mg/mL of the fusion protein.
  • the aqueous formulation comprises at least 10 mM histidine. In some embodiments, the aqueous formulation comprises at least 15 mM, 20 mM, 25 mM, 30 mM, or 35 mM histidine. In some embodiments, the aqueous formulation comprises 10-50 mM histidine, or 10-40 mM, 15-35 mM, 20-30 mM, 22-28 mM, or 24-26 mM histidine. In some embodiments, the aqueous formulation comprises 25 mM histidine.
  • the aqueous formulation comprises at least 100 mM trehalose. In some embodiments, the aqueous formulation comprises at least 100 mM, 150 mM, 200 mM, 250 mM or 300 mM trehalose. In some embodiments, the aqueous formulation comprises 100-400 mM trehalose, or 150-350 mM, 200-300 mM, 220-280 mM, 240-260 mM, or 245-255 mM trehalose. In some embodiments, the aqueous formulation comprises 250 mM trehalose.
  • the aqueous formulation comprises a polysorbate, such as polysorbate 20 or polysorbate 80. In some embodiments, the aqueous formulation comprises at least 0.005 (w/v) % polysorbate. In some embodiments, the aqueous formulation comprises at least 0.01 (w/v) % polysorbate, or at least 0.015 (w/v) % polysorbate, at least 0.02 (w/v) % polysorbate, or at least 0.025 (w/v) % polysorbate. In some embodiments, the aqueous formulation comprises 0.01 (w/v)% to 0.03 (w/v)% polysorbate 80.
  • the aqueous formulation comprises 0.015 (w/v) %-0.025 (w/v) % polysorbate 80. In some embodiments, the aqueous formulation comprises 0.018 (w/v)% to 0.022 (w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.019 (w/v)% to 0.021 (w/v)% polysorbate 80. In some embodiments, the aqueous formulation comprises 0.02 (w/v) % polysorbate 80.
  • the pH of the aqueous formulation is 7.0 or higher. In some embodiments, the pH of the aqueous formulation is 7.1 or higher, 7.2 or higher, 7.3 or higher, 7.4 or higher, 7.5 or higher, or 7.6 or higher. In some embodiments, the pH of the aqueous formulation is 7.0-8.2, or 7.1-8.1, 7.2-8.0, 7.3-7.9, 7.4-7.8, 7.5-7.7, or 7.55-7.65. In some embodiments, the pH of the aqueous formulation is 7.6.
  • the aqueous formulation comprises at least 25 mg/mL of the fusion protein, 24-26 mM histidine, 240-260 mM trehalose, and 0.015 (w/v)%-0.025 (w/v)% Polysorbate 80, and its pH is 7.4-7.8.
  • the aqueous formulation consists of at least 25 mg/mL of the fusion protein, 24-26 mM histidine, 240-260 mM trehalose, and 0.015 (w/v)%-0.025 (w/v)% Polysorbate 80 composition, and its pH is 7.4-7.8.
  • the aqueous formulation comprises at least 25 mg/mL of the fusion protein, 25 mM histidine, 250 mM trehalose, and 0.02 (w/v) % polysorbate 80, and has a pH of 7.6.
  • the aqueous formulation consists of 30 mg/mL of the fusion protein, 25 mM histidine, 250 mM trehalose, and 0.02 (w/v) % polysorbate 80, and has a pH of 7.6.
  • the aqueous formulation further comprises tonicity agents (or osmotic pressure regulators, stabilizers), surfactants, antioxidants, preservatives, or mixtures thereof.
  • tonicity agents or osmotic pressure regulators, stabilizers
  • surfactants or antioxidants, preservatives, or mixtures thereof.
  • the aqueous formulation further includes a tonicity agent (or osmotic pressure regulator, stabilizer).
  • a tonicity agent refers to a pharmaceutically acceptable agent used to adjust the tonicity of a formulation. Isotonicity generally relates to the osmotic pressure of a solution relative to a solution, usually relative to human serum.
  • the formulation may be hypotonic, isotonic or hypertonic.
  • the formulation is isotonic.
  • Isotonic formulations are liquids or liquids reconstituted from solid forms (eg, from lyophilized forms) and refer to solutions that have the same tonicity as some other solutions to which they are compared, such as physiological saline solutions and serum. Suitable isotonic agents include, but are not limited to, sodium chloride, potassium chloride, glycerol, mannitol, and any components from amino acids, sugars, as defined herein, and combinations thereof.
  • the aqueous formulation further includes a surfactant.
  • surfactant refers to a pharmaceutically acceptable organic substance with an amphiphilic structure; that is, it consists of groups with opposite solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group .
  • surfactants can be classified into anionic, cationic and nonionic surfactants depending on the charge of the surface active moiety. Surfactants are commonly used as wetting agents, emulsifiers, solubilizers, and dispersants for various pharmaceutical compositions and biomaterial formulations.
  • the amount of surfactant is described as a percentage (w/v%) expressed as a weight/volume percentage.
  • Suitable pharmaceutically acceptable surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid ester (Tween), polyoxyethylene alkyl ether, alkyl phenyl polyoxyethylene ether (Triton-X), Group of polyoxyethylene-polyoxypropylene copolymers (Poloxamers, Pluronic) or sodium dodecyl sulfate (SDS).
  • Polyoxyethylene sorbitan-fatty acid esters include polysorbate 20 (sold under the trademark Tween 20 TM ) and polysorbate 80 (sold under the trademark Tween 80 TM ).
  • Polyethylene-polypropylene copolymers include under the name Or those sold by Poloxamer 188 TM .
  • Polyoxyethylene alkyl ethers include those sold under the trademark Brij TM .
  • Alkylphenol ethoxylates include those sold under the tradename Triton-X.
  • the aqueous formulation further includes an antioxidant.
  • Antioxidant refers to a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidant. Oxidative reactions can generate free radicals that initiate chain reactions that destabilize the protein therapeutic and ultimately affect the activity of the product. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by oxidizing themselves.
  • antioxidants are typically reducing agents, chelating agents and oxygen scavengers such as citrates, EDTA, DPTA, thiols, ascorbic acid or polyphenols.
  • antioxidants include ascorbic acid (AA, E300), thiosulfate, methionine, tocopherol (E306), propyl gallate (PG, E310), tert-butyl hydroquinone (TBHQ) ), butylated hydroxyanisole (BHA, E320) and butylated hydroxytoluene (BHT, E321).
  • the aqueous formulation further includes a preservative.
  • Preservatives are natural or synthetic chemicals that are added to products such as food, pharmaceuticals, coatings, biological samples, wood, etc. to prevent degradation by microbial growth or by undesired chemical changes. Preservative additives can be used alone or in combination with other preservative methods. Preservatives can be antimicrobial preservatives, which inhibit the growth of bacteria and fungi, or antioxidants such as oxygen scavengers, which inhibit the oxidation of components.
  • Common antimicrobial preservatives include benzalkonium chloride, benzoic acid, cholorohexidine, glycerol, benzoic acid, potassium sorbate, thimerosal, sulfites (sulfur dioxide, sodium bisulfite, potassium bisulfite, etc. ) and disodium EDTA.
  • Other preservatives include those commonly used for parenteral proteins, such as benzyl alcohol, phenol, m-cresol, chlorobutanol or methylparaben.
  • the aqueous formulation does not contain these excipients.
  • the aqueous formulation does not contain more than 10 mM (or does not contain more than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM) of other amino acid salts other than histidine salts, such as arginine acid, lysine, asparagine, glutamine, glycine, or their salts. In some embodiments, the aqueous formulation does not contain other amino acid salts other than histidine salts, such as arginine, lysine, asparagine, glutamine, glycine, or their salts.
  • the aqueous formulation contains no more than 10 mM (or no more than 5 mM, 2 mM, 1 mM, 0.1 mM, or 0.01 mM) of sugars other than trehalose, such as sucrose. In some embodiments, the aqueous formulation does not contain sugars other than trehalose, such as sucrose.
  • the present specification also describes corresponding dry formulations, such as lyophilized formulations.
  • the dry formulation is obtainable by lyophilizing the aqueous formulation described herein.
  • the dry formulation with the addition of a suitable amount of water, can result in an aqueous formulation as described herein.
  • the formulations described in this application can be used to treat a variety of corresponding diseases.
  • the gp130 dimers of the present invention have higher binding affinity than native soluble gp130 and thus have a stronger ability to inhibit IL-6 signaling.
  • IL-6 signaling is associated with numerous diseases, including those briefly described below, and others commonly known to those of skill in the art.
  • Chronic inflammation such as Crohn's disease (CD), ulcerative colitis (UC), rheumatoid arthritis (RA), or psoriasis
  • CD Crohn's disease
  • UC ulcerative colitis
  • RA rheumatoid arthritis
  • psoriasis is histologically associated with the presence of monocytes such as macrophages and lymphocytes , persists in tissues after acquisition for resolution of the acute inflammatory phase.
  • monocytes such as macrophages and lymphocytes
  • IL-6 appears to have deleterious effects favoring monocyte accumulation at sites of injury by inducing sustained MCP-I secretion, vascular proliferation, and anti-apoptotic functions on T cells.
  • IBD Inflammatory bowel disease
  • ie CD or UC is a chronic inflammation that occurs in the gut of susceptible individuals that is not thought to be associated with a specific pathogen.
  • Alterations in the epithelial mucosal barrier accompanied by increased intestinal permeability lead to enhanced exposure of the mucosal immune system to luminal antigens, which leads to inappropriate activation of the patient's intestinal immune system.
  • Uncontrolled activation of mucosal CD4+ T-lymphocytes is accompanied by a continuous excessive release of proinflammatory cytokines, inducing pathogenic gastrointestinal inflammation and tissue damage.
  • the main activated immune cells involved in the pathogenesis of IBD are intestinal T cells and macrophages.
  • IL-6 has been shown in humans as a central cytokine in IBD. Compared with controls, (3) and UC patients have been found to produce elevated levels of IL-6, and IL-6 levels correlate with clinical activity. It was also found that (3) patients had elevated levels of SIL-6R, and thus elevated levels of IL-6/sIL-6R complex in serum. Lamina intestinal mononuclear cells obtained from surgical colon specimens of CD and UC patients showed that both CD4+ T cells and macrophages produced increased amounts of IL-6 compared to controls. SIL-6R was found to be released by shedding from the surface of macrophages and monocytes, with an accompanying increase in production associated with elevated levels of IL-6.
  • mucosal T cells show strong evidence for IL-6 trans-signaling, with concomitant activation of STAT3, bcl-2 and bcl-xl. Blockade of IL-6 trans-signaling induced T cell apoptosis, suggesting that the IL-6/sIL-6R system mediates T cell resistance to apoptosis in CD.
  • the formulations of the present invention can treat IL-6 mediated disorders.
  • Disorders mediated by IL-6 include inflammatory diseases or cancer.
  • the polypeptides and compositions described herein can be administered to subjects with inflammatory diseases, such as juvenile idiopathic arthritis, Crohn's disease, colitis (eg, colitis not associated with IBD, including radiation colitis, diverticulitis, ischemic colitis, infectious colitis, celiac disease, autoimmune colitis or colitis caused by allergies affecting the colon), dermatitis, psoriasis, uveitis, diverticulitis inflammation, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, nephritis, Parkinson's disease, ulcerative colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma and various cardiovascular diseases such as atherosclerosis and vasculitis.
  • the inflammatory disease is selected from the group
  • the inflammatory disease or IL-6 mediated disorder is inflammatory bowel disease, preferably wherein the treatment induces remission of the inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease or ulcerative colitis, preferably wherein the treatment maintains remission of the inflammatory bowel disease.
  • the inflammatory disease or IL-6 mediated disorder is rheumatoid arthritis, psoriasis, uveitis or atherosclerosis.
  • the inflammatory disease or IL-6 mediated disorder is colitis unrelated to inflammatory bowel disease, preferably wherein the colitis is radiation colitis, diverticulitis, ischemic colitis, infectious colitis, lipid Diarrhea, autoimmune colitis, or colitis caused by allergies affecting the colon.
  • the inflammatory disease or IL-6 mediated disorder is selected from Crohn's disease, ulcerative colitis, rheumatoid arthritis and psoriasis, more preferably from Crohn's disease and ulcerative colitis.
  • treatment can include remission of the condition, maintenance of remission of the condition, or both.
  • cancer including but not limited to multiple myeloma, plasma cell leukemia, renal cell carcinoma, Kaposi's sarcoma, colorectal cancer, gastric cancer, black melanoma, leukemia, lymphoma, glioma, glioblastoma multiforme, lung cancer (including but not limited to non-small cell lung cancer (NSCLC; adenocarcinoma and squamous cell carcinoma)), non-Hodgkin lymphoma tumor, Hodgkin's disease, plasmacytoma, sarcoma, thymoma, breast cancer, prostate cancer, hepatocellular cancer, bladder cancer, uterine cancer, pancreatic cancer, esophageal cancer, brain cancer, head and neck cancer, ovarian cancer, cervical cancer , testicular cancer, gastric cancer, esophageal cancer, liver cancer, acute lymphoblastic leukemia (ALL), T-
  • ALL acute lymphoblastic leukemia
  • Additional embodiments of the present disclosure provide methods of treating, reducing the severity of, or preventing a disease selected from the group consisting of sepsis, bone resorption (osteoporosis), cachexia, cancer-related fatigue, psoriasis, systemic Juvenile idiopathic arthritis, systemic lupus fibrinopathy (SLE), mesangial proliferative glomerulonephritis, hypergammaglobulinemia, Castleman's disease, IgM gammopathy, cardiac myxoma and autoimmune insulin-dependent diabetes mellitus.
  • a disease selected from the group consisting of sepsis, bone resorption (osteorosis), cachexia, cancer-related fatigue, psoriasis, systemic Juvenile idiopathic arthritis, systemic lupus fibrinopathy (SLE), mesangial proliferative glomerulonephritis, hypergammaglobulinemia, Castleman's disease, IgM
  • the cDNA encoding SEQ ID NO: 1 can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the amino acid sequence of the antibody chain.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).
  • Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as those derived from retrovirus LTR, cytomegalovirus (CMV) (such as CMV promoter/enhancer), Simian virus 40 (SV40) (e.g. SV40 promoter/enhancer), adenovirus (e.g. adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters (e.g.
  • the host cells may be mammalian, insect, plant, bacterial or yeast cells, preferably the cells are mammalian cells such as Chinese Hamster Ovary (CHO) cells.
  • CHO cells are (CHO)/dhfr - cells obtained from the European Collection of Cell Cultures (ECACC, no. 9406067).
  • the host cell is a CHO cell, and the nucleic acid encoding the polypeptide is codon-optimized for use in the CHO cell.
  • Another aspect of the present disclosure includes fusion proteins produced by the methods disclosed herein.
  • the dimer has the characteristics described herein (eg, % galactose-alpha-1,3-galactose per mole of polypeptide, sialylation).
  • the dimers produced by the method can be used to prepare suitable compositions.
  • the fusion protein molecule thus produced contains no more than 6 galactose-alpha-1,3-galactose, or no more than 3, 2, or 1 galactose-alpha-1,3-galactose.
  • the fusion protein molecules so produced comprise glycans, wherein on average at least 52% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average at least 54% of the glycans comprise one or more sialic acid residues. In some embodiments, the fusion protein comprises glycans, wherein on average 52-65% of the glycans comprise one or more sialic acid residues.
  • IM001 protein is obtained from CHO-K1 engineered cells expressing gp130-Fc fusion protein gene after cell culture, isolation and purification.
  • IM001 The cDNA sequence of IM001 was expressed in a CHO cell expression system.
  • the presence of the IgG1 Cys-Pro-Pro-Cys sequence in the Fc region results in the dimerization of two identical gp130-Fc subunits through sulfhydryl residues on the Fc region, which together form IM001.
  • the pilot manufacturing and purification process for the IM001 drug substance is as follows. Cells were recovered from WCB vials and gradually expanded using protein-free medium prior to inoculation into production bioreactors. After cell culture is complete, cells and cell debris are removed by culture filtration. Purification consisted of three column chromatography steps, a concentration and diafiltration step, and two specific virus clearance steps (virus inactivation treatment and nanofiltration, removal of enveloped and non-enveloped viruses). After concentration and diafiltration, excipients are added to formulate the drug substance. The formulated IM001 was filtered through a 0.22 ⁇ m filter into the container.
  • This example also attempts to screen the most stable pH/buffer system for the IM001 protein.
  • the IM001 stock solution was exchanged to 20mM acetate (pH4.5, pH5.0, pH5.5), citrate (pH5.0), histidine-aspartate (pH5.0, pH5.5), histidine (pH5.5, pH6.0, pH6.5, pH7.0, pH8.0) and phosphate (pH7.0).
  • the protein concentration was adjusted to around 30 mg/mL, and then filtered through a 0.22 ⁇ m PVDF membrane filter.
  • the filtered samples were bottled and sealed. All manipulations were performed in a biosafety hood.
  • One of the bottles was used as T0, and the appropriate stability conditions were selected according to the results of TODSC and HT-DLS, and the rest of the samples were stored under these conditions.
  • the results are shown in Table 6.
  • the results of reducing SDS-PAGE showed that the purity of p10 (His at pH 7.0), p11 (His at pH 8.0) and p12 (Phosphate at pH 7.0) did not decrease after being stored at 30°C for 2 weeks, and the remaining samples were all Different degrees of decrease occurred, and the decrease range was between 10.8% and 50.0%; the purity of non-reducing SDS-PAGE showed different degrees of decrease, among which p10 (His at pH 7.0), p11 (His at pH 8.0) and p12 (pH 8.0) Phosphate of 7.0) decreased less than the rest of the samples, which decreased by 14.6% to 65.7%.
  • the research purpose of this example is to screen suitable excipient stabilizers on the basis of pH/buffer system screening.
  • the 25mM histidine buffer system with pH 8.0 was used as the excipient to screen the main evaluation buffer system.
  • the glycine buffer system of pH 7.5 and the Tris buffer system of pH 7.5 were added.
  • the IM001 stock solution was changed to 25mM histidine buffer system (pH 8.0), 25mM glycine buffer system (pH7.5) and 25mM Tris buffer system (pH7.5) by ultrafiltration centrifugation.
  • the research purpose of this example is to screen high-concentration freeze-dried formulations.
  • the protein concentration of IM001 increased to 30 mg/mL, and the purity of IM001 decreased significantly with the prolonged storage time at 25°C or higher. Therefore, the formulation design of the lyophilized preparation was carried out, and the scheme is shown in Table 14. Change the IM001 stock solution to 25mM histidine buffer system (pH 8.0), add different auxiliary material mother liquid and surfactant mother liquid respectively according to the experimental plan, and adjust the protein content and add different kinds of sugar mother liquid and surfactant mother liquid respectively, And adjust the protein content to 30mg/mL.
  • the results of the moisture measurement of the freeze-dried product are shown in Table 16.
  • the moisture content of different prescriptions is all less than 3%, which has reached the moisture content standard of the freeze-dried product, and the specific moisture content is between 1.1 and 1.6%.
  • the moisture content of F7 is slightly higher, which is 1.63%.
  • Tg' glass transition temperature
  • Tc freeze-drying collapse temperature
  • the 5 freeze-dried products investigated all had good appearance and were white loose blocks.
  • the moisture content measurement results are between 1.1% and 1.6%, all of which are less than 3%, and the moisture content is qualified.
  • the reconstituted solutions were light yellow, slightly opalescent, and free of visible particles.
  • the Tg' of F7 (containing 4% mannitol and 2% sucrose) is -33.2°C, and the Tg' of other formulations are about -26 to -27°C. There was no significant difference in MFI results between different prescriptions.
  • the SEC results showed that all the formulations investigated except F7 had better stability after freeze-drying.
  • the SEC main peak (containing 4% mannitol and 2% sucrose) decreased more than the other formulations and was less stable.
  • the screening results of excipients and surfactants showed that the stability of pH 8.0 histidine buffer system was stronger than that of glycine and Tris buffer system, the protective effect of sucrose and trehalose was similar, better than that of mannitol, and the protein of 15 mg/mL
  • the concentration stability is better than the system of 30mg/mL, among which the aqueous formulation of F9 formula (15mg/mL protein, 25mM His, 250mM Sucrose, 0.02% PS80, pH 7.6) has the best stability.
  • the results of the screening experiment of freeze-dried preparations showed that the stability of freeze-dried preparations was significantly improved compared with the solution formulations.
  • the F7 formulation (30mg/mL protein, 25mM His pH 8.0, 4% Mannitol, 2% Sucrose, 0.02% PS80) performed slightly worse, and the other formulations showed excellent stability.

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Abstract

本发明提供一种水性制剂，包含两个单链gp130-Fc融合蛋白的二聚体，组氨酸盐，海藻糖和聚山梨酯80。所述水性制剂可用于治疗炎性疾病或IL-6介导的病症。

Description

包含可溶性gp130二聚体的制剂和使用方法 技术领域
本发明属于生物医药研究领域，具体涉及一种包含gp130二聚体的制剂，以及用来治疗各种IL-6介导的病症，包括炎性疾病和癌症的用途。
背景技术
糖蛋白130(也称为gp130，IL6ST，IL6-beta或CD130)是一种跨膜蛋白。它形成IL-6受体家族中I型细胞因子受体的一个亚基。它对于细胞因子参与后的信号转导很重要。在结构上，gp130在其细胞外部分由五个纤连蛋白III型结构域和一个免疫球蛋白样C2型结构域组成。
IL-6受体家族的成员都通过与gp130形成复合体以进行信号转导。例如，IL-6与IL-6受体结合。然后，这两种蛋白质的复合物与gp130缔合。然后，由3种蛋白质组成的复合物均二聚化，形成可以产生下游信号的六聚体复合物。
IL-6是由造血细胞和非造血细胞产生的多效细胞因子，例如响应于感染和组织损伤。IL-6通过两种主要信号转导途径发挥其多重生物活性，-种是通过主要存在于肝细胞和某些白细胞上的膜结合IL-6R的所谓经典配体-受体途径，以及一种是通过源自膜结合IL-6R的蛋白水解切割或源自选择性剪接的循环sIL-6R(soluble IL-6R，或称可溶性IL-6R)的跨信号转导途径(反式信号途径)。
在经典途径中，IL-6直接与有限范围细胞类型的表面上的膜结合的IL-6R结合。IL-6/IL-6R复合物与信号转导性gp130受体蛋白质的预先形成的二聚体缔合，引起gp130同二聚体的空间变化，并从而引发细胞内信号转导级联。经典信号转导负责急性炎症防御机制和关键的生理学IL-6功能，如肠上皮细胞的生长和再生信号。IL-6R和gp130的细胞外结构域可通过翻译选择性剪接的mRNA而产生，无膜-锚定结构域，从而产生sIL-6R和gp130变体。
IL-6/sIL-6R复合物的活性通常由循环中存在的高水平可溶性sgp130 (soluble gp130)控制，其与膜结合的gp130有效竞争。本发明中的gp130二聚体比天然sgp130具有更高结合亲和力并因此具有更强的抑制IL-6信号转导的能力。本发明的制剂让该gp130二聚体能够在生产、运输和运用中更稳定。
发明内容
为了克服现有技术中所存在的问题，本发明描述了一种包含gp130二聚体(或者“包含gp130的融合蛋白”，或者简称“融合蛋白”)的制剂，以及用来治疗各种IL-6介导的病症，包括炎性疾病和癌症的用途。该制剂包含一个组氨酸盐缓冲体系，在pH7.6左右，有很高的稳定性，并且可以安全地以各种剂量施用于人类。
在一种实施方式中，本说明书描述了一种水性制剂(也可称作液体制剂)和一种冻干制剂，其包含一种融合蛋白、20-30mM组氨酸盐、220-280mM海藻糖和0.01(w/v)％-0.03(w/v)％聚山梨酯80，而且其pH为7.0-8.2，所述融合蛋白包含两个氨基酸序列为SEQ ID NO：1的单体，且由多个二硫键相连接。在一些实施方式中，其包含至少10mg/mL，15mg/mL，20mg/mL，25mg/mL，或者至少30mg/mL该融合蛋白。
在一些实施方式中，所述水性制剂的pH为7.4-7.8。在一些实施方式中，所述水性制剂的pH为7.6。
在一些实施方式中，所述水性制剂包含24-26mM组氨酸盐。在一些实施方式中，所述水性制剂包含25mM组氨酸盐。
在一些实施方式中，所述水性制剂包含240-260mM海藻糖。在一些实施方式中，所述水性制剂包含250mM海藻糖。
在一些实施方式中，所述水性制剂包含0.015(w/v)％-0.025(w/v)％聚山梨酯80。在一些实施方式中，所述水性制剂包含0.02(w/v)％聚山梨酯80。
在一些实施方式中，所述水性制剂进一步包括张度剂(或称为渗透压调节剂或稳定剂)、表面活性剂、抗氧化剂、防腐剂、或其混合物。
在一些实施方式中，所述每个融合蛋白分子包含不多于6个半乳糖-α-1，3-半乳糖。在一些实施方式中，所述每个融合蛋白分子包含不多于3个，2个，或者1个半乳糖-α-1，3-半乳糖。
在一些实施方式中，所述融合蛋白包含聚糖，其中平均至少52％所述聚糖包 含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均至少54％所述聚糖包含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均52-65％所述聚糖包含一个或多个唾液酸残基。
在一种优选的实施方式中，所述的水性制剂包含至少25mg/mL该融合蛋白、24-26mM组氨酸盐、240-260mM海藻糖和0.015(w/v)％-0.025(w/v)％聚山梨酯80，而且其pH为7.6-7.8。
在一种优选的实施方式中，所述的水性制剂包含30mg/mL该融合蛋白、25mM组氨酸盐、250mM海藻糖和0.02(w/v)％聚山梨酯80，而且其pH为7.6。
在一些实施方式中，所述的水性制剂不包含高于10mM(或者不包含高于5mM，2mM，1mM，0.1mM，或者0.01mM)的除组氨酸盐外的其他氨基酸盐，或者，优选的，完全不包含其他任何氨基酸盐。
在一些实施方式中，所述的水性制剂不包含高于10mM(或者不包含高于5mM，2mM，1mM，0.1mM，或者0.01mM)的除海藻糖外的其他糖，或者，优选的，完全不包含其他任何糖。
在一些实施方式中，所述的水性制剂，用于治疗人类中的炎性疾病或IL-6介导的病症。在一些实施方式中，其中所述炎性疾病或IL-6介导的病症是炎症性肠病，优选地其中所述治疗诱导炎症性肠病的缓解。在一些实施方式中，其中所述炎症性肠病是克罗恩氏病或溃疡性结肠炎，优选地其中所述治疗维持炎症性肠病的缓解。在一些实施方式中，其中所述炎性疾病或IL-6介导的病症是类风湿性关节炎、牛皮癣、葡萄膜炎或动脉粥样硬化。在一些实施方式中，其中所述炎性疾病或IL-6介导的病症是与炎症性肠病无关的结肠炎，优选地其中所述结肠炎是放射性结肠炎、憩室结肠炎、缺血性结肠炎、感染性结肠炎、脂泻病、自身免疫性结肠炎、或由影响结肠的过敏引起的结肠炎。
本说明书也捕述了一种干制剂，其可以通过冻干本文所述的任何一种水性制剂获得，或者加入水后可以生成本文所述的任何一种水性制剂。
附图说明
图1，pH/缓冲体系筛选DSC结果。
图2，pH/缓冲体系筛选HT-DLS结果。
图3，辅料及表面活性剂筛选DSC结果
图4，不同处方冻干品外观
具体实施方式
定义
所有数字标号(例如pH、温度、时间、浓度和分子量，包括范围)均为以0.1或10％的增量(1)或(一)变化的近似值。应了解，尽管并非始终明确地陈述，所有数字标号之前均有术语“约”。还应了解，尽管并非始终明确地陈述，本文所述的试剂仅为示例性的并且其相等物为本领域中已知的。
术语“蛋白”和“多肽”可互换使用并且以其最广泛意义指代两种或更多种亚单位氨基酸、氨基酸类似物或肽模拟物的化合物。所述亚单位可由肽键键联。在另一实施方案中，所述亚单位可由例如酯、醚等其它键键联。蛋白或肽必须含有至少两种氨基酸并且关于可构成蛋白或肽的序列的氨基酸的最大数目并无限制。如本文所用，术语“氨基酸”指代天然和/或非天然或合成氨基酸，包括甘氨酸和D和L光学异构体、氨基酸类似物和肽模拟物。天然存在氨基酸的单字母和三字母缩写列于下文。
“组合物”意图意指活性剂和另一惰性(例如，可检测试剂或标记)或活性化合物或组合物(如佐剂)的组合。“药物组合物”意图包括活性剂与惰性或活性载体的组合，从而产生适用于体外、体内或离体诊断或治疗用途的组合物。
“水性制剂”是指使用水作为溶剂的液体制剂。在一种实施方式中，水性制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
如本文所用，术语“缓冲液”表示药学上可接受的赋形剂，其稳定化药物制剂的pH。合适缓冲液为本领域中众所周知的并且可发现于文献中。药学上可接受的缓冲液包含但不限于tri s缓冲液、精氨酸缓冲液、组氨酸缓冲液、柠檬酸盐缓冲液、丁二酸盐缓冲液和磷酸盐缓冲液。与所用的缓冲液无关，pH可用本领域中已知的酸或碱调节，例如丁二酸、盐酸、乙酸、磷酸、硫酸和柠檬酸、丁二酸盐、柠檬酸盐、tris碱、组氨酸、组氨酸HCl、氢氧化钠和氢氧化钾。合适缓冲液包括而不限于组氨酸缓冲液、2-吗啉代乙烷磺酸(MES)、二甲胂酸盐、磷酸盐、乙酸盐、丁二酸盐和柠檬酸盐。所述缓冲液的浓度可在约4mM与约60mM，或替代地约4mM至约40mM，或替代地约5mM至约25mM之间。
如本文中所使用的，术语“治疗”、“疗法”和“处理”是指逆转如本文所述的疾病或病症或其一个或多个症状、减轻疾病或病症或其一个或多个症状、延迟疾病或病症或其一个或多个症状的发作或抑制疾病或病症或其一个或多个症状的进展。在一些实施方式中，治疗可以在一个或多个症状发展之后施用。在其它实施方式中，治疗可以在没有症状的情况下施用。例如，治疗可以在症状发作之前施用于易感个体(例如考虑到症状史和/或考虑到遗传或其他易感因素)。症状解除后也可继续治疗，例如预防或延迟复发。
本发明各个主题中的专有名词或术语的解释以及实施例均可通用，不再重复赘述。
制剂
IM001是一种包含两个单链gp130-Fc融合蛋白的二聚体。它可以用来治疗各种IL-6介导的病症，包括炎性疾病和癌症。和其他蛋白质药物相似，IM001的可溶性、稳定性和活性受其环境的影响。所以，开发出一个合适的制剂，包括合适的缓冲体系，并不容易。
本发明人制备了12种pH/缓冲体系处方(表2)、9种辅料及表面活性剂水性制剂筛选处方(表7)，30℃考察2周稳定性，通过DSC，DLS，外观，蛋白浓度，pH，SEC-HPLC，SDS(还原/非还原)方法对他们进行比较。发现pH≤6.5，有明显可见异物产生，蛋白也很不稳定，而其在pH 8.0(偏碱性)的缓冲体系中更稳定。此外，对于相同pH 8.0缓冲体系，组氨酸缓冲体系的稳定性强于甘氨酸和Tris缓冲体系。比较有趣的是，IM001蛋白加到缓冲体系后pH会有漂移，pH 8.0组氨酸缓冲体系，加入蛋白后pH漂移为7.6。
在对各种水性制剂处方考察后，本申请发明人发现，在蔗糖和聚山梨酯80保护效果下，15mg/mL的蛋白浓度稳定性最佳。不过，可惜的是，当蛋白浓度达到30mg/mL时，其稳定性不够。
这个结果比较令人意外，因为很多含有Fc片段的蛋白，比如抗体，可以较容易地开发出浓度更高的稳定的水性制剂。不受任何特定理论的束缚，本申请发明人认为这是因为本融合蛋白分子特性，高温或高浓度容易不稳定，加上等电点原因可选择pH缓冲体系也有限，相对一般单抗等生物药分子制剂开发挑战性更 大。
为了提高高浓度制剂处方稳定性，本申请发明人制备了5个冻干制剂筛选处方(表14)，通过25℃和40℃条件下稳定性考察，实验结果显示，相同高浓度30mg/mL，冻干品的稳定性比溶液处方均有明显的改善。有趣的是冻干处方(30mg/mL 25mM His，250mM Trehalose，0.02％PS80，pH 7.6)在不同温度和各种测试中都表现出很好的结果，尤其是高温40℃条件下2个月基本没有变化，这样的结果是非常意外的，因为一般来说，同一个制剂并不见得在各种测试中都有优势。因此将其都作为IM001蛋白的冻干制剂处方。而且，这个冻干制剂处方所用的糖(海藻糖)也不同于水性制剂处方中的优选方案(蔗糖)。
根据这些测试结果，本申请提供了一种适用于IM001的水性制剂和冻干制剂，其包含一种融合蛋白、组氨酸盐、海藻糖和聚山梨酯。在一些实施方式中，该水性制剂冻干后可以形成冻干制剂。在一些实施方式中，该冻干制剂在加入合适的水后，可以生产该水性制剂。这样的水性制剂也可以注射给病人治疗适应的疾病。
如前所述，这里的融合蛋白(IM001)包含两个氨基酸序列为SEQ ID NO：1的单体，由多个二硫键相连接。在一些实施方式中，所述每个融合蛋白分子包含不多于6个半乳糖-α-1，3-半乳糖。在一些实施方式中，所述每个融合蛋白分子包含不多于3个，2个，或者1个半乳糖-α-1，3-半乳糖。在一些实施方式中，所述融合蛋白包含聚糖，其中平均至少52％所述聚糖包含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均至少54％所述聚糖包含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均52-65％所述聚糖包含一个或多个唾液酸残基。
在一些实施方式中，所述水性制剂包含至少10mg/mL，15mg/mL，20mg/mL，25mg/mL，或者至少30mg/mL该融合蛋白。在一些实施方式中，所述水性制剂包含10-60mg/mL，15-45mg/mL，20-40mg/mL，25-35mg/mL，或者30mg/mL该融合蛋白。
在一些实施方式中，所述水性制剂包含至少10mM组氨酸盐。在一些实施方式中，所述水性制剂包含至少15mM，20mM，25mM，30mM，或者35mM组氨酸盐。在一些实施方式中，所述水性制剂包含10-50mM组氨酸盐，或者10-40mM，15-35mM，20-30mM，22-28mM，或者24-26mM组氨酸盐。在一些实施方式中， 所述水性制剂包含25mM组氨酸盐。
在一些实施方式中，所述水性制剂包含至少100mM海藻糖。在一些实施方式中，所述水性制剂包含至少100mM，150mM，200mM，250mM或者300mM海藻糖。在一些实施方式中，所述水性制剂包含100-400mM海藻糖，或者150-350mM，200-300mM，220-280mM，240-260mM，或者245-255mM海藻糖。在一些实施方式中，所述水性制剂包含250mM海藻糖。
在一些实施方式中，所述水性制剂包含聚山梨酯，比如聚山梨酯20或者聚山梨酯80。在一些实施方式中，所述水性制剂包含至少0.005(w/v)％聚山梨酯。在一些实施方式中，所述水性制剂包含至少0.01(w/v)％聚山梨酯，或者至少0.015(w/v)％聚山梨酯，至少0.02(w/v)％聚山梨酯，或者至少0.025(w/v)％聚山梨酯。在一些实施方式中，所述水性制剂包含0.01(w/v)％-0.03(w/v)％聚山梨酯80。在一些实施方式中，所述水性制剂包含0.015(w/v)％-0.025(w/v)％聚山梨酯80。在一些实施方式中，所述水性制剂包含0.018(w/v)％-0.022(w/v)％聚山梨酯80。在一些实施方式中，所述水性制剂包含0.019(w/v)％-0.021(w/v)％聚山梨酯80。在一些实施方式中，所述水性制剂包含0.02(w/v)％聚山梨酯80。
在一些实施方式中，所述水性制剂的pH为7.0或者更高。在一些实施方式中，所述水性制剂的pH为7.1或者更高，7.2或者更高，7.3或者更高，7.4或者更高，7.5或者更高，或者7.6或更高。在一些实施方式中，所述水性制剂的pH为7.0-8.2，或者7.1-8.1，7.2-8.0，7.3-7.9，7.4-7.8，7.5-7.7，或者7.55-7.65。在一些实施方式中，所述水性制剂的pH为7.6。
在一个示范性实施方式中，所述水性制剂包含至少25mg/mL该融合蛋白、24-26mM组氨酸盐、240-260mM海藻糖和0.015(w/v)％-0.025(w/v)％聚山梨酯80，而且其pH为7.4-7.8。在一个示范性实施方式中，所述水性制剂由至少25mg/mL该融合蛋白、24-26mM组氨酸盐、240-260mM海藻糖和0.015(w/v)％-0.025(w/v)％聚山梨酯80组成，而且其pH为7.4-7.8。
在一个示范性实施方式中，所述水性制剂包含至少25mg/mL该融合蛋白、25mM组氨酸盐、250mM海藻糖和0.02(w/v)％聚山梨酯80，而且其pH为7.6。在一个示范性实施方式中，所述水性制剂由30mg/mL该融合蛋白、25mM组氨酸盐、250mM海藻糖和0.02(w/v)％聚山梨酯80组成，而且其pH为7.6。
在一些实施方式中，所述水性制剂进一步包括张度剂(或叫渗透压调节剂， 稳定剂)、表面活性剂、抗氧化剂、防腐剂、或者其混合物。
在一些实施方式中，所述水性制剂进一步包括张度剂(或叫渗透压调节剂，稳定剂)。如本文所用，术语“张度剂”表示用于调节制剂的张度的药学上可接受的试剂。等张性一般涉及相对于溶液，通常相对于人类血清的溶液的渗透压。制剂可为张力减退的、等张的或张力亢进的。一方面，所述制剂为等张的。等张制剂为液体或从固体形式(例如从冻干形式)复原的液体并且表示与一些其它与其进行比较的溶液(如生理盐溶液和血清)具有相同张度的溶液。合适等张剂包括但不限于氯化钠、氯化钾、甘油、甘露醇和来自如本文所定义的氨基酸、糖的任何组分以及其组合。
在一些实施方式中，所述水性制剂进一步包括表面活性剂。如本文所用，术语“表面活性剂”指代具有两亲结构的药学上可接受的有机物质；即，其由具有相反溶解倾向的基团构成，一般为油溶性烃链和水溶性离子基团。表面活性剂可视表面活性部分的电荷分类为阴离子性、阳离子性和非离子性表面活性剂。表面活性剂通常用作用于多种药物组合物和生物材料制剂的湿润剂、乳化剂、增溶剂和分散剂。在本文所述的药物制剂的一些实施方案中，表面活性剂的量是描述为以重量/体积百分比表示的百分率(w/v％)。合适的药学上可接受的表面活性剂包括但不限于聚氧乙烯山梨醇酐脂肪酸酯(Tween)、聚氧乙稀烧基醚、烧基苯基聚氧乙稀醚(Triton-X)、聚氧乙稀-聚氧丙烯共聚物(泊洛沙姆，Pluronic)或十二烷基硫酸钠(SDS)的组。聚氧乙烯山梨醇酐-脂肪酸酯包括聚山梨醇酯20(以商标Tween 20 TM出售)和聚山梨醇酯80(以商标Tween 80 TM出售)。聚乙烯-聚丙烯共聚物包括以名称
Figure PCTCN2021143870-appb-000001
或泊洛沙姆188 TM出售的那些。聚氧乙烯烷基醚包括以商标Bri j TM出售的那些。烷基苯酚聚氧乙烯醚包括以商品名Triton-X出售的那些。
在一些实施方式中，所述水性制剂进一步包括抗氧化剂。“抗氧化剂”指代能够减慢或防止其它分子的氧化的分子。氧化是从物质转移电子至氧化剂的化学反应。氧化反应可产生自由基，所述自由基开始使蛋白治疗剂不稳定并且最终影响产物活性的链反应。抗氧化剂通过去除自由基中间物终止这些链反应，并且通过氧化自身来抑制其它氧化反应。因此，抗氧化剂通常为还原剂、螯合剂和氧清除剂，如柠檬酸盐、EDTA、DPTA、硫醇、抗坏血酸或多酚。抗氧化剂的非限制性实例包括抗坏血酸(AA，E300)、硫代硫酸盐、甲硫氨酸、生育酚(E306)、没食子酸丙酯(PG，E310)、叔丁基对苯二酚(TBHQ)、丁基化羟基茴香醚(BHA， E320)和丁基化羟基甲苯(BHT，E321)。
在一些实施方式中，所述水性制剂进一步包括防腐剂。“防腐剂”为天然或合成化学品，其添加至如食物、医药品、涂料、生物样品、木材等产品中以防止因微生物生长或因不期望的化学改变而降解。防腐剂添加剂可单独或联合其它防腐方法使用。防腐剂可为抗微生物防腐剂，其抑制细菌和真菌的生长，或如氧清除剂的抗氧化剂，其抑制组分氧化。常见抗微生物防腐剂包括苯扎氯铵、苯甲酸、氯己啶(cholorohexidine)、甘油、苯酸、山梨酸钾、硫柳萊、亚硫酸盐(二氧化硫、亚硫酸氢钠、亚硫酸氢钾等)和EDTA二钠。其它防腐剂包括常用于肠胃外蛋白的那些，如苯甲醇、苯酚、间甲酚、氯代丁醇或对羟基苯甲酸甲酯。
从实施例可见，某些常用的生物药物制剂辅料，比如甘氨酸、Tris、甘露醇等，并没有对形成优秀的水性或者冻干制剂带来帮助。所以，在一些实施方式中，所述水性制剂并不包含这些辅料。
在一些实施方式中，所述的水性制剂不包含高于10mM(或者不包含高于5mM，2mM，1mM，0.1mM，或者0.01mM)的除组氨酸盐外的其他氨基酸盐，比如精氨酸、赖氨酸、天冬酰胺、谷氨酰胺、甘氨酸、或者他们的盐。在一些实施方式中，所述的水性制剂不包含除组氨酸盐外的其他氨基酸盐，比如精氨酸、赖氨酸、天冬酰胺、谷氨酰胺、甘氨酸、或者他们的盐。
在一些实施方式中，所述的水性制剂不包含高于10mM(或者不包含高于5mM，2mM，1mM，0.1mM，或者0.01mM)的除海藻糖外的其他糖，比如蔗糖。在一些实施方式中，所述的水性制剂不包含除海藻糖外的其他糖，比如蔗糖。
在一些实施方式中，本说明书也描述了相应的干制剂，比如冻干制剂。在一些实施方式中，该干制剂是可以通过冻干本说明书所述的水性制剂获得。在一些实施方式中，该干制剂在加入合适量的水后，可以产生本说明书所述的水性制剂。
用途
本申请描述的制剂可以用来治疗各种相应的疾病。本发明中的gp130二聚体比天然可溶性gp130具有更高结合亲和力并因此具有更强的抑制IL-6信号转导的能力。IL-6信号转导和众多疾病相关，包括下面简单描述的，和本领域 技术人员普通了解的其他疾病。
慢性炎症，如克罗恩氏病(CD)、溃疡性结肠炎(UC)、类风湿性关节炎(RA)或牛皮癣，在组织学上与单核细胞如巨噬细胞和淋巴细胞的存在有关，在获得用于解决急性炎症期后持续存在于组织中。在慢性炎性疾病模型中，通过在T细胞上诱导持续的MCP-I分泌、血管增生和抗凋亡功能，IL-6似乎具有有利于损伤部位的单核细胞积累的有害作用。
炎症性肠病(IBD)，即CD或UC，是易感个体肠道中发生的慢性炎症，其被认为与特定病原体无关。上皮粘膜屏障中的改变伴随着肠道通透性增加导致粘膜免疫系统增强暴露于肠腔抗原，这导致患者肠道免疫系统的不适当活化。粘膜CD4+T-淋巴细胞的不受控制的激活伴随着促炎细胞因子的连续过度释放，诱导致病性胃肠道炎症和组织损伤。有一个共识，即参与IBD发病机制的主要活化免疫细胞是肠T细胞和巨噬细胞。
IL-6在人体中显示为IBD中的中枢细胞因子。与对照组相比，已经发现(3)和UC患者产生升高水平的IL-6，IL-6水平与临床活动相关。也发现(3)患者的SIL-6R水平升高，并因此血清中IL-6/sIL-6R复合物的水平升高。从CD和UC患者的手术结肠标本获得的固有层固有单核细胞显示，与对照相比，CD4+T细胞和巨噬细胞都产生增加的IL-6量。发现SIL-6R通过从巨噬细胞和单核细胞的表面脱落而释放，伴随着与IL-6水平升高相关的生产增加。在CD患者中，粘膜T细胞显示出对IL-6跨信号转导的强烈证据，伴随有STAT3、bcl-2和bcl-xl的活化。IL-6跨信号转导的阻断引起T细胞凋亡，表明IL-6/sIL-6R系统在CD中介导T细胞对细胞凋亡的抗性。
因此，在IBD患者中，导致炎症持续存在的固有层中的促炎CD4+T细胞的获得性积累严重依赖于抗凋亡IL-6/sIL-6R跨信号转导。据信，通过作用于IL-6/sIL-6R复合物，本文公开的多肽可用于治疗CD和其它炎性疾病。
因此，本发明的制剂可以治疗IL-6介导的病症。IL-6介导的病症包括炎性疾病或癌症。在这方面，本文所述的多肽和组合物可以施用于患有炎性疾病的受试者，如幼年特发性关节炎、克罗恩氏病、结肠炎(例如与IBD无关的结肠炎，包括放射性结肠炎、憩室结肠炎、缺血性结肠炎、感染性结肠炎、脂泻病、自身免疫性结肠炎或由影响结肠的过敏引起的结肠炎)、皮炎、牛皮癣、葡萄膜炎、憩室炎、肝炎，肠易激综合征(IBS)、红斑狼疮、肾炎、帕金森病、溃疡性结肠炎、 多发性硬化症(MS)、阿尔茨海默病、关节炎、类风湿关节炎、哮喘和各种心血管疾病如动脉粥样硬化和血管炎。在某些实施方式中，炎症性疾病选自于由糖尿病、痛风、冷冻蛋白相关周期性综合征和慢性阻塞性肺疾病组成的组。
优选地，炎性疾病或IL-6介导的病症是炎症性肠病，优选其中治疗诱导炎症性肠病的缓解。优选地，炎症性肠病是克罗恩氏病或溃疡性结肠炎，优选其中治疗维持炎症性肠病的缓解。优选地，炎性疾病或IL-6介导的病症是类风湿性关节炎、牛皮癣、葡萄膜炎或动脉粥样硬化。优选地，炎性疾病或IL-6介导的病症是与炎症性肠病无关的结肠炎，优选其中结肠炎是放射性结肠炎、憩室结肠炎、缺血性结肠炎、感染性结肠炎、脂泻病、自身免疫性结肠炎或由影响结肠的过敏引起的结肠炎。优选地，炎性疾病或IL-6介导的病症选自克罗恩氏病、溃疡性结肠炎、类风湿性关节炎和牛皮癣，更优选选自克罗恩氏病和溃疡性结肠炎。
对于诸如炎症性肠病等炎性疾病，治疗可以包括病症的缓解、病症缓解的维持或两者。
其他实施方式提供了治疗癌症、降低癌症的严重性或预防癌症的方法，癌症包括但不限于多发性骨髓瘤、浆细胞白血病、肾细胞癌、卡波西氏肉瘤、结肠直肠癌、胃癌、黑素瘤、白血病、淋巴瘤、神经胶质瘤、多形性成胶质细胞瘤、肺癌(包括但不限于非小细胞肺癌(NSCLC；腺癌和鳞状细胞癌))、非霍奇金淋巴瘤、霍奇金病、浆细胞瘤、肉瘤、胸腺瘤、乳腺癌、前列腺癌、肝细胞癌、膀胱癌、子宫癌、胰腺癌、食管癌、脑癌、头颈癌、卵巢癌、子宫颈癌、睾丸癌、胃癌、食管癌、肝癌、急性淋巴细胞白血病(ALL)、T-ALL、急性髓性白血病(AML)、慢性粒性白血病(CML)和慢性淋巴细胞性白血病(CLL)、唾液癌或其它癌症。
本公开的另外的实施方式提供了治疗疾病、降低疾病的严重性或预防疾病的方法，该疾病选自于由败血症、骨吸收(骨质疏松症)、恶病质、癌症相关疲劳、牛皮癣、全身性青少年特发性关节炎、系统性纤斑狼疮(SLE)、肾小球膜增生性肾小球肾炎、高丙种球蛋白血症、卡斯尔曼氏病、IgM丙种球蛋白病、心脏粘液瘤和自身免疫性胰岛素依赖性糖尿病组成的组。
生产方法
本公开的进一步方面提供了生产该制剂的方式。编码SEQ ID NO：1的cDNA可以被克隆到载体中，以便信号肽与抗体链的氨基酸序列的氨基末端框内连接。 信号肽可以是免疫球蛋白信号肽或异源信号肽(即来自非免疫球蛋白的信号肽)。
表达载体的设计，包括调控序列的选择，可取决于诸如待转化的宿主细胞的选择、所需蛋白质的表达水平等因素。用于哺乳动物宿主细胞表达的调控序列包括在哺乳动物细胞中引导高水平蛋白质表达的病毒元素，如源自逆转录病毒LTR、细胞巨化病毒(CMV)(诸如CMV启动子/增强子)、猿猴病毒40(SV40)储如SV40启动子/增强子)、腺病毒(例如腺病毒主要晚期启动子(AdMLP))、多瘤和强哺乳动物启动子(如天然免疫球蛋白和肌动蛋白启动子)的启动子和/或增强子。宿主细胞可以是哺乳动物、昆虫、植物、细菌或酵母细胞，优选细胞是哺乳动物细胞，如中国仓鼠卵巢(CHO)细胞。示例性CHO细胞是从欧洲细胞培养物保藏中心(ECACC，编号9406067)获得的(CHO)/dhfr -细胞。优选地，宿主细胞是CHO细胞，并且编码多肽的核酸被密码子优化以用于CHO细胞中。
本公开的另一方面包括通过本文公开的方法产生的融合蛋白。优选地，二聚体具有本文描述的特征(例如，每摩尔多肽的％半乳糖-α-1，3-半乳糖，唾液酸化)。由所述方法生产的二聚体可以被用来制备适合的组合物。这样生产的融合蛋白分子包含不多于6个半乳糖-α-1，3-半乳糖，或者不多于3个，2个，或者1个半乳糖-α-1，3-半乳糖。
这样生产的融合蛋白分子包含聚糖，其中平均至少52％所述聚糖包含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均至少54％所述聚糖包含一个或多个唾液酸残基。在一些实施方式中，所述融合蛋白包含聚糖，其中平均52-65％所述聚糖包含一个或多个唾液酸残基。
实施例1
IM001蛋白由表达gp130-Fc融合蛋白基因的CHO-K1工程细胞，经细胞培养、分离和纯化后获得。
表1.IM001单链的氨基酸序列
Figure PCTCN2021143870-appb-000002
Figure PCTCN2021143870-appb-000003
IM001的cDNA序列用CHO细胞表达系统中表达。Fc区中IgGl Cys-Pro-Pro-Cys序列的存在导致两个相同的gp130-Fc亚单元通过Fc区上的巯基残基而二聚化，它们一起形成IM001。
IM001药物物质的试点性制造和纯化过程如下。在接种到生产生物反应器中之前，使用无蛋白质培养基，从WCB小瓶中复苏细胞并逐渐扩大。细胞培养完成后，通过培养物过滤除去细胞和细胞碎片。纯化由三个色谱柱层析步骤、一个浓缩和渗滤步骤以及两个特定的病毒清除步骤(病毒灭活处理和纳滤、去除包膜和非包膜病毒)组成。浓缩和渗滤后，加入赋形剂以配制药物物质。将配制的IM001通过0.22μm滤膜过滤到容器中。
本实施例也尝试筛选IM001蛋白最稳定的pH/缓冲体系。
通过透析方法将IM001原液分别交换到20mM醋酸盐(pH4.5，pH5.0，pH5.5)，柠檬酸盐(pH5.0)，组氨酸-天冬氨酸盐(pH5.0，pH5.5)，组氨酸(pH5.5，pH6.0，pH6.5，pH7.0，pH8.0)和磷酸盐(pH7.0)。将蛋白质浓度调节到30mg/mL左右，然后用0.22μm PVDF膜过滤器进行过滤。滤后样品进行装瓶，密封。所有操作均在生物安全罩中进行。其中一瓶作为T0，根据T0DSC及HT-DLS结果选择合适的稳定性考察条件，并将其余样品按该条件储存，每瓶按表2规定的时间点取样分析。
表2.IM001 pH/缓冲体系筛选研究方案
Figure PCTCN2021143870-appb-000004
Figure PCTCN2021143870-appb-000005
1.1.DSC及HT-DLS结果
结果详见表3，图1-图2。所有处方Tm-onset为40.9-43.0℃，Tagg Onset除处方160.6℃和处方1165.7℃外，其他处方Tagg Onset均在45℃左右。
表3.IM001 pH/缓冲体系筛选DSC及HT-DLS结果
Figure PCTCN2021143870-appb-000006
1.2.外观、pH和蛋白浓度结果
结果详见表4。外观结果显示T0时除处方1和处方11无可见异物外，所有处方均出现可见异物，30℃下存放1周和两周后，所有处方均出现可见异物。pH和蛋白浓度结果显示30℃存放2周所有处方均无明显变化。
表4.IM001 pH/缓冲体系筛选外观、pH和蛋白浓度结果
Figure PCTCN2021143870-appb-000007
Figure PCTCN2021143870-appb-000008
注：SY(淡黄色)/SO(微乳光)/OL(乳光)，FP(无可见异物)/PO(有可见异物)/+(少量可见异物)/++(大量可见异物)
1.3.SEC纯度结果
结果详见表5，结果显示在30℃存放1-2周后，所有处方SEC纯度出现不同程度的下降，其中处方11下降的程度最低(约13％)，其他缓冲体系下降幅度范围是19.3％-72％。
表5.IM001 pH/缓冲体系筛选SEC纯度结果
Figure PCTCN2021143870-appb-000009
1.4.SDS-PAGE(还原/非还原)结果
结果详见表6，还原SDS-PAGE结果显示在30℃存放2周后，p10(pH 7.0 的His)、p11(pH 8.0的His)和p12(pH 7.0的Phosphate)纯度未下降，其余样品均发生不同程度的下降，下降幅度在10.8％～50.0％之间；非还原SDS-PAGE纯度均出现不同程度的下降，其中p10(pH 7.0的His)、p11(pH 8.0的His)和p12(pH 7.0的Phosphate)的下降幅度小于其余样品，其余样品的下降幅度为14.6％～65.7％。
表6.IM001 pH/缓冲体系筛选SDS-PAGE(还原/非还原)结果
Figure PCTCN2021143870-appb-000010
本研究通过DSC，DLS，外观，蛋白浓度，pH，SEC-HPLC，SDS-PAGE(还原/非还原)方法筛选IM001蛋白最稳定的pH/缓冲体系。DSC和DLS结果中显示IM001分子的Tm Onset和Tagg Onset随pH值升高呈上升趋势，其中p11(pH 8.0的His)的Tm Onset和Tagg Onset高于其余样品。在30℃条件下考察2周后，外观，SEC-HPLC，SDS-PAGE(还原/非还原)结果显示IM001在低pH环境中相对不稳定，处方11(组氨酸盐缓冲体系，pH8.0，含蛋白pH7.6)在每一项测试中都相对优于其他处方。
实施例2
本实施例的研究目的是在pH/缓冲体系筛选基础上筛选合适辅料稳定剂。
根据pH/缓冲体系筛选实验结果，将pH 8.0的25mM组氨酸缓冲体系作为辅料筛选主要评估的缓冲体系。此外增加考察pH7.5的甘氨酸缓冲体系和pH 7.5的Tris缓冲体系。通过超滤离心的方式将IM001原液换到25mM组氨酸缓冲体系(pH 8.0)、25mM甘氨酸缓冲体系(pH7.5)和25mM Tris缓冲体系(pH7.5)中，根据实验方案分别添加不同的辅料母液和表面活性剂母液，并调节蛋白质含量分别至30mg/mL与15mg/mL左右，最终制备含有不同辅料和表面活性剂的9个处方。在生物安全柜中分别使用0.22μm PVDF膜过滤后，分装至西林瓶中，加塞密封。开始不同条件的实验考察，具体方案见表7，表中处方pH为未加蛋白前的缓冲液pH，因IM001蛋白加到缓冲体系中pH有漂移现象，实际处方pH见测定结果。
Figure PCTCN2021143870-appb-000011
Figure PCTCN2021143870-appb-000012
Figure PCTCN2021143870-appb-000013
Figure PCTCN2021143870-appb-000014
Figure PCTCN2021143870-appb-000015
Figure PCTCN2021143870-appb-000016
Figure PCTCN2021143870-appb-000017
Figure PCTCN2021143870-appb-000018
Figure PCTCN2021143870-appb-000019
实施例3
本实施例的研究目的是筛选高浓度冻干制剂处方。
根据水性制剂处方筛选结果，IM001蛋白浓度提高到30mg/mL，25℃或更高温度随着放置时间延长，纯度有明显下降趋势。因此进行了冻干制剂的处方设计，方案见表14。将IM001原液换液到25mM组氨酸缓冲体系(pH 8.0)中，根据实验方案分别添加不同的辅料母液和表面活性剂母液，并调节蛋白质含量分别添加不同种类糖的母液和表面活性剂母液，并调节蛋白质含量至30mg/mL。将制备好的5个处方在生物安全柜中分别使用0.22μm的PVDF膜过滤后，用移液器取5mL药液至已清洗灭菌的20R西林瓶中，并及时加塞、轧盖和贴签。然后冻干开始不同条件的稳定性考察，具体方案见表15。
表14冻干制剂筛选处方列表
Figure PCTCN2021143870-appb-000020
表15冻干品稳定性实验方案
Figure PCTCN2021143870-appb-000021
其中X＝外观，渗透压，pH，蛋白浓度，SEC-HPLC；
Y＝MFI；
Z＝水分含量，Tc，Tg’
3.1冻干品外观观察
冻干品外观见图4所示，所有处方冻干后外观均为白色疏松块状，其中F7(含有4％甘露醇和2％蔗糖)性状较其他冻干品更加疏松。
3.2冻干品水分含量测定
冻干品水分测定的结果见表16，不同处方的水分含量均小于3％，达到了冻干品水分含量标准，具体水分含量在1.1～1.6％之间。其中F7水分含量稍高，为1.63％。
表16冻干品水分含量测定
处方编号 样品编号 水分含量
F2 2226-20210509 1.39％
F7 2226-20210510 1.63％
F15 2226-20210511 1.12％
F16 2226-20210512 1.17％
F17 2226-20210513 1.17％
3.3冻干处方冷冻溶液玻璃化转变温度(Tg’)和冻干塌陷温度(Tc)测定
冻干处方冷冻溶液玻璃化转变温度(Tg’)和冻干塌陷温度(Tc)测定结果见表17，含有蔗糖的处方F2，F16，F17的Tg’均在-27℃左右，含有海藻糖的处方F7的Tg’略高，为-25.9℃，而含有4％甘露醇和2％蔗糖处方F7的Tg’最低，为-33.2℃。另外，我们测定了F2和F15两个处方的Tc，分别为-23.8℃和-26.7℃。
表17冻干溶液玻璃化转变温度(Tg’)和冻干塌陷温度(Tc)测定结果
处方编号 样品编号 Tg’ Tc
F2 2226-20210509 -27.2℃ -23.8℃
F7 2226-20210510 -33.2℃ NA
F15 2226-20210511 -25.9℃ -26.7℃
F16 2226-20210512 -27.7℃ NA
F17 2226-20210513 -27.9℃ NA
3.4冻干品复溶溶液外观检测
根据冻干品冻干前后的质量变化差异，计算得到复溶加水体积，加水将冻干品复溶，在复溶完全后观测复溶溶液的外观，结果见表18所示。所有样品，包括T0，放置在加速条件(25℃)以及高温条件(40℃)的样品，复溶后的溶 液均为淡黄色，微乳光，均无可见颗粒。
表18冻干品复溶溶液外观测定结果
Figure PCTCN2021143870-appb-000022
注：C无色(colorless)/SY淡黄色(slightly yellow)/Y黄色(yellow)；
CL澄明(Clear liquid)/微乳光SO(slightly opalescent liquid)/乳光O(opalescent liquid)；
FP无可见颗粒(free of visible particles)/PO少量颗粒(a few visible particles)
Figure PCTCN2021143870-appb-000023
3.6冻干品复溶溶液不溶性微粒检测(MFI)
冻干品复溶溶液的不溶性微粒检测(MFI)结果汇总见表20，不同处方不溶性微粒数量没有明显差异，且25℃放置1个月或40℃放置2个月后的样品，复溶后不溶性微粒数相比T0杆品没有明显上升。
表20冻干品复溶溶液不溶性微粒检测(MFI)结果
Figure PCTCN2021143870-appb-000024
* 1，2这两个样品可能是被污染了，从表中数据可以看出F2的25C-3M，40C-1M以及40C-2M样品的不溶性微粒明显低于T0样品，而对于F15也可以发现40C-1M，40C-2M样品不溶性微粒数明显低于25C-3M样品。
3.7冻干品复溶溶液纯度(SEC-HPLC)检测
SEC-HPLC检测结果汇总见表21，所有处方在加速条件(25℃)和高温条件(40℃)下放置2周至3个月，除了F7SEC纯度有微小下降，其余SEC纯度基本没有变化，说明冻干品的稳定性相比液体制剂有了显著提升。其中F7处方(含有4％甘露醇和2％蔗糖)稳定性相比其他处方较差，可能与退火后甘露醇结晶，蛋白失去糖的保护作用有关，其余处方SEC主峰含量基本不变，均有良好的稳定性。
表21冻干品复溶溶液纯度(SEC-HPLC)检测结果
Figure PCTCN2021143870-appb-000025
Figure PCTCN2021143870-appb-000026
3.8冻干制剂筛选实验总结
所考察的5个冻干品都有较好的外观，为白色疏松块状。水分含量测定结果在1.1～1.6％之间，均小于3％，水分含量合格。放置在加速条件(25℃)和高温条件(40℃)2周至3个月后，复溶溶液均为淡黄色，微乳光，且无可见颗粒。F7(含有4％甘露醇和2％蔗糖)的Tg’为-33.2℃，其余处方的Tg’为-26～-27℃左右。MFI结果不同处方间无明显差异。SEC结果显示除F7外所有考察处方冻干后稳定性较好，放置在加速条件(25℃)下3个月和高温条件(40℃)下2个月，其主峰均无明显下降，F7处方(含有4％甘露醇和2％蔗糖)的SEC主峰相比其他处方下降更多，其稳定性更差。
总结
缓冲体系筛选结果显示，pH 8.0His缓冲盐(处方pH 7.6)体系的稳定性最佳。
辅料和表面活性剂筛选结果显示，pH 8.0组氨酸缓冲体系的稳定性强于甘氨酸和Tris缓冲体系，蔗糖和海藻糖的保护效果相近，优于甘露醇的保护效果，并且15mg/mL的蛋白浓度稳定性优于30mg/mL的体系，其中F9处方(15mg/mL蛋白，25mM His，250mM Sucrose，0.02％PS80，pH 7.6)的水性制剂稳定性最佳。
冻干制剂筛选实验结果显示，冻干品的稳定性相比溶液处方均有明显的改善。其中F7处方(30mg/mL蛋白，25mM His pH 8.0，4％Mannitol，2％Sucrose，0.02％PS80)的表现稍差，其余处方均表现出优良的稳定性。
根据以上实验结果，我们选择了F15处方(30mg/mL蛋白，25mM His，250mM Trehalose，0.02％PS80，pH 7.6)作为IM001蛋白的冻干制剂处方。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (27)

  1. 一种水性制剂，其包含一种融合蛋白、20-30mM组氨酸盐、220-280mM海藻糖和0.01(w/v)％-0.03(w/v)％聚山梨酯80，且其pH为7.0-8.2，所述融合蛋白包含两个氨基酸序列为SEQ ID NO:1的单体，并由多个二硫键相连接。
  2. 根据权利要求1所述的水性制剂，其中所述水性制剂的pH为7.4-7.8。
  3. 根据权利要求2所述的水性制剂，其中所述水性制剂的pH为7.6。
  4. 根据权利要求1所述的水性制剂，其中所述水性制剂包含24-26mM组氨酸盐。
  5. 根据权利要求4所述的水性制剂，其中所述水性制剂包含25mM组氨酸盐。
  6. 根据权利要求1所述的水性制剂，其中所述水性制剂包含240-260mM海藻糖。
  7. 根据权利要求6所述的水性制剂，其中所述水性制剂包含250mM海藻糖。
  8. 根据权利要求1所述的水性制剂，其中所述水性制剂包含0.015(w/v)％-0.025(w/v)％聚山梨酯80。
  9. 根据权利要求8所述的水性制剂，其中所述水性制剂包含0.02(w/v)％聚山梨酯80。
  10. 根据权利要求1所述的水性制剂，其进一步包括张度剂、表面活性剂、抗氧化剂、防腐剂、或其混合物。
  11. 根据权利要求1所述的水性制剂，其中，所述每个融合蛋白分子包含不多于6个半乳糖-α-1,3-半乳糖。
  12. 根据权利要求11所述的水性制剂，其中，所述每个融合蛋白分子包含不多于3个半乳糖-α-1,3-半乳糖。
  13. 根据权利要求11所述的水性制剂，其中，所述融合蛋白包含聚糖，其中平均至少52％所述聚糖包含一个或多个唾液酸残基。
  14. 根据权利要求1所述的水性制剂，其中包含至少10mg/mL该融合蛋白。
  15. 根据权利要求14所述的水性制剂，其中包含至少25mg/mL该融合蛋白。
  16. 根据权利要求1所述的水性制剂，其中包含至少25mg/mL该融合蛋白、24-26mM组氨酸盐、240-260mM海藻糖和0.015(w/v)％-0.025(w/v)％聚山梨酯80，而且其pH为7.4-7.8。
  17. 根据权利要求16所述的水性制剂，其中包含30mg/mL该融合蛋白、25mM组氨酸盐、250mM海藻糖和0.02(w/v)％聚山梨酯80，而且其pH为7.6。
  18. 根据权利要求1-17中任一项所述的水性制剂，其中不包含高于10mM的除组氨酸盐外的其他氨基酸盐，或者，优选的，不包含其他氨基酸盐。
  19. 根据权利要求18所述的水性制剂，其中不包含高于10mM的除海藻糖外的其他糖，或者，优选的，不包括除海藻糖外的其他糖。
  20. 根据权利要求19所述的水性制剂，其由30mg/mL该融合蛋白、25mM组氨酸盐、250mM海藻糖和0.02(w/v)％聚山梨酯80组成，而且其pH为7.6。
  21. 根据权利要求1-20中任一项所述的水性制剂，用于治疗人类中的炎性疾病或IL-6介导的病症。
  22. 根据权利要求21所述的用于使用的水性制剂，其中所述炎性疾病或IL-6介导的病症是炎症性肠病，优选地其中所述治疗诱导炎症性肠病的缓解。
  23. 根据权利要求21所述的用于使用的水性制剂，其中所述炎症性肠病是克罗恩氏病或溃疡性结肠炎，优选地其中所述治疗维持炎症性肠病的缓解。
  24. 根据权利要求21所述的用于使用的水性制剂，其中所述炎性疾病或IL-6介导的病症是类风湿性关节炎、牛皮癣、葡萄膜炎或动脉粥样硬化。
  25. 根据权利要求21所述的用于使用的水性制剂，其中所述炎性疾病或IL-6介导的病症是与炎症性肠病无关的结肠炎，优选地其中所述结肠炎是放射性结肠炎、憩室结肠炎、缺血性结肠炎、感染性结肠炎、脂泻病、自身免疫性结肠炎、或由影响结肠的过敏引起的结肠炎。
  26. 一种干制剂，其可以通过冻干权利要求1-20中任一项所述的水性制剂获得。
  27. 一种干制剂，其加入水后可以生成权利要求1-20中任一项所述的水性制剂。
PCT/CN2021/143870 2020-12-31 2021-12-31 包含可溶性gp130二聚体的制剂和使用方法 WO2022143999A1 (zh)

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