WO2019024783A1 - COMPOSITION D'ANTICORPS ANTI-TNF-α, ET SON APPLICATION - Google Patents

COMPOSITION D'ANTICORPS ANTI-TNF-α, ET SON APPLICATION Download PDF

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WO2019024783A1
WO2019024783A1 PCT/CN2018/097445 CN2018097445W WO2019024783A1 WO 2019024783 A1 WO2019024783 A1 WO 2019024783A1 CN 2018097445 W CN2018097445 W CN 2018097445W WO 2019024783 A1 WO2019024783 A1 WO 2019024783A1
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tnf
antibody
concentration
composition
preparation
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PCT/CN2018/097445
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Chinese (zh)
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林键
王盛武
岳海涛
裴树军
李胜峰
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百奥泰生物科技(广州)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to the novel pharmaceutical preparations, and in particular to an antibody composition against TNF- ⁇ and uses thereof.
  • RA Rheumatoid arthritis
  • TNF- ⁇ tumor necrosis factor- ⁇
  • interleukins such as IL-1, IL-6, IL-8, etc.
  • pro-inflammatory cytokines such as TNF- ⁇ inhibitors, anti-IL-6R antibodies, etc.
  • the main mechanisms of action of these biological agents are: (1) a monoclonal antibody against a cytokine or a receptor thereof; (2) a soluble receptor antagonist, that is, a cytokine membrane receptor that does not include a transmembrane component and an intracellular functional region. Receptor antagonists bind to free cytokines and inhibit binding of the latter to cell surface receptors.
  • Soluble receptor antagonists have a short half-life and can be prolonged by the addition of certain structures such as Fc receptors of human IgG or polyethylene glycol (PEG); (3) receptor antagonistic proteins, which are biologically inactive. Protein, which competes with cytokines for binding to membrane receptors on the cell surface. Receptor antagonistic proteins must be combined with more than 90% of cell surface receptors to be considered effective.
  • TNF- ⁇ is one of the most important pro-inflammatory cytokines in many cytokines of RA inflammatory response, and it plays an important role in the development of RA, local inflammation and tissue damage (Fe ldmann M, Brennan FM, Foxwell BMJ, et al. The role of TNF and IL-1 in rheumatoid art hritis. CurrDir Au toimmun, 2001; 3: 188-199.).
  • the levels of TNF- ⁇ and TNF- ⁇ receptor (TNFR) in RA were significantly elevated in serum, synovium, and synovial fluid, especially in active patients with severe disease.
  • Serum TNF- ⁇ levels were positively correlated with joint damage score, erythrocyte sedimentation rate (ESR), and anemia. TNF- ⁇ is also associated with weight loss and disease recurrence in RA patients. TNF- ⁇ can act on a variety of cells, such as promoting macrophage secretion of inflammatory cytokines and chemokines, and promoting inflammation.
  • TNF- ⁇ The main functions of TNF- ⁇ are: (1) induce endothelial cells to express adhesion molecules and vascular endothelial growth factor (VEGF), promote adhesion and penetration of leukocytes and vascular endothelium, leading to local inflammatory reaction; (2) acting on hepatocytes, producing C-reactive protein (CRP); (3) In RA, TNF- ⁇ can act on osteoclasts, synoviocytes and chondrocytes, respectively, resulting in the activation of these cells, producing metalloproteinases, collagenases, and basal membrane lytic enzymes ( Stromelysin) and prostaglandin E2 (PGE2) further destroy cartilage causing bone erosion, joint inflammation and cartilage destruction; (4) TNF- ⁇ also promotes the production of IL-1 by synoviocytes, macrophages, fibroblasts and chondrocytes IL-8 and TNF- ⁇ themselves aggravate tissue damage.
  • VEGF vascular endothelial growth factor
  • TNF- ⁇ inhibition of TNF- ⁇ is important for controlling the condition of RA and improving prognosis (Daniel Tracey, Lars Klareskog, Eric H. Sssso, etal. Tumor necrosis factor antagon ist mechanisms of action A comprehensive review. Pharmacology and Therapeutics, 2008; 244-279).
  • TNF- ⁇ inhibitors There are five TNF- ⁇ inhibitors approved by the US FDA: soluble receptor antagonist - Etanercept, human and mouse chimeric antibody - Infliximab, all-human monoclonal antibody Adalimumab, a fully human monoclonal antibody, Golimumab and a polyethylene glycol humanized Fab' fragment, Certolizumab pegol.
  • Etanercept is a recombinant human type II tumor necrosis factor receptor-antibody fusion protein composed of a type II TNF receptor (p75) and a Fc portion of IgG1;
  • Infliximab is composed of a human Ig stable region and a murine Ig variable region.
  • TNF inhibitors are injection site reactions, infections, tumors, lymphoproliferative disorders, neurodegenerative lesions, and lupus-like syndrome (DJ Shealy, S. Visvanathan. Anti-TNF Antibodies: Lessons from the Past, Roadmap For the Future. Handbook of Experimental Pharmacology 2008; 181: 101-129.
  • TNF- ⁇ inhibitors are intravenous and subcutaneous.
  • Subcutaneous preparations are more convenient for patients to use than intravenous preparations, and the injection time is greatly shortened. It is not required to be injected by a professional physician, and the patient can inject the medicine after receiving the training.
  • factors such as formulation ingredients and pH tend to cause discomfort and even obvious pain when the patient is injected, which may lead to the patient not taking the medicine according to the doctor's advice, especially in the case of non-fatal diseases such as inflammatory diseases. Laursen et al.
  • citrate buffer formulations caused more pain in patients than the histidine buffer formulation (Laursen T, Hansen B) , Fisker S. Pain Perception after Subcutaneous Injections of Media Containing Different Buffers. Basic & Clinical Pharmacology & Toxicology, 2006; 98(2): 218-221).
  • the present invention provides an antibody composition against TNF- ⁇ , wherein the solvent comprises water, the solute comprises an anti-TNF- ⁇ antibody; the antibody composition has a pH of 4.0 to 8.0, and the pH is The liquid component of the antibody composition is pressurized with carbon dioxide.
  • the solute further comprises a stabilizer, a surfactant;
  • the anti-TNF- ⁇ antibody comprises a recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406.
  • the antibody composition for TNF-[alpha], including the type and amount of solute comprises:
  • the concentration of the anti-TNF- ⁇ antibody is 20.0-130.0 mg/ml
  • the stabilizer comprises one or two of 20.0-60.0 mg/ml polyol and 70.0-100.0 mg/ml sugar;
  • the surfactant comprises 0.5-2.0 mg / ml polysorbate 80;
  • the antibody composition has a pH of from 4.0 to 8.0, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 10-300 mmHg.
  • the antibody composition for TNF-[alpha] including the type and amount of solute, satisfies one or more of the following preferred conditions:
  • the concentration of the anti-TNF- ⁇ antibody is 20.0-110.0 mg/ml
  • the type of the polyol includes mannitol or sorbitol, the concentration of the polyol is 30.0-50.0 mg/ml; the kind of the saccharide includes sucrose or trehalose, and the concentration of the saccharide is 80.0-90.0 mg. /ml;
  • the concentration of the polysorbate 80 is 0.8-1.5 mg / ml
  • the antibody composition has a pH of from 4.9 to 5.7, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 100-300 mmHg.
  • the antibody composition for TNF-[alpha] including the type and amount of solute, satisfies one or more of the following preferred conditions:
  • the concentration of the anti-TNF- ⁇ antibody is 50 mg/ml or 100 mg/ml;
  • the type of the polyol includes mannitol or sorbitol, the concentration of the polyol is 42 mg/ml, and the type of the sugar includes sucrose or trehalose, and the concentration of the saccharide is 84 mg/ml;
  • the concentration of the polysorbate 80 is 1.0 mg / ml
  • the antibody composition has a pH of 5.0 to 5.4, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 220 mmHg.
  • the antibody composition for TNF-[alpha], the species of the solute and its content comprises:
  • the concentration of the anti-TNF- ⁇ antibody is 100.0 mg/ml
  • the stabilizer is mannitol at a concentration of 42.0 mg/ml.
  • the surfactant is polysorbate 80, and the concentration is 1.0 mg/ml.
  • the antibody composition has a pH of 5.0 to 5.4, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 220 mmHg.
  • the antibody composition for TNF-[alpha], including the type and amount of solute comprises:
  • the concentration of the anti-TNF- ⁇ antibody is 70.0-130.0 mg/ml
  • the stabilizer includes one or two of 3.0-15.0 mg/ml polyol and 5.0-8.0 mg/ml sodium chloride.
  • the surfactant comprises 3.0-10 mg / ml polysorbate 80;
  • the antibody composition has a pH of from 4.0 to 6.0, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 20-80 mmHg.
  • the antibody composition for TNF-[alpha] including the type and amount of solute, satisfies one or more of the following preferred conditions:
  • the concentration of the anti-TNF- ⁇ antibody is 90.0-110.0 mg/ml
  • the type of the stabilizer includes mannitol, sodium chloride, the concentration of the mannitol is 8.0-12.0 mg/ml, and the concentration of the sodium chloride is 6.0-7.0 mg/ml;
  • the surfactant comprises 4.0-8.0 mg/ml polysorbate 80;
  • the antibody composition has a pH of from 4.8 to 5.5, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 40-60 mmHg.
  • the antibody composition for TNF-[alpha] including the type and amount of solute, satisfies one or more of the following preferred conditions:
  • the concentration of the anti-TNF- ⁇ antibody is 100 mg/ml
  • the concentration of mannitol is 10.0 mg/ml, and the concentration of the sodium chloride is 6.5 mg/ml;
  • the concentration of the polysorbate 80 is 6.0 mg / ml
  • the antibody composition has a pH of 5.0 to 5.4, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 50 mmHg.
  • the antibody composition for TNF-[alpha] is as follows:
  • the concentration of the anti-TNF- ⁇ antibody is 100.0 mg/ml
  • the stabilizer comprises 10.0 mg/ml of mannitol and 6.5 mg/ml of sodium chloride.
  • the surfactant is polysorbate 80, and the concentration is 6 mg/ml.
  • the antibody composition has a pH of 5.0 to 5.4, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 50 mmHg.
  • the invention provides an antibody preparation for TNF-[alpha] comprising a closed container, and said antibody composition for TNF-[alpha] in a closed container, said composition having a pH of from 4.0 to 8.0.
  • the closed container comprises a pressure can, a vial, an injection needle, and the like.
  • the present invention provides a use according to the antibody composition for TNF- ⁇ in the preparation of a therapeutic agent for a TNF- ⁇ -causing disorder.
  • the therapeutic agent comprises a subcutaneous injection formulation.
  • the present invention has the following beneficial effects:
  • the antibody composition of the present invention does not contain a common buffer system, and replaces the buffer system by filling carbon dioxide and maintains a stable pH of the preparation, thereby reducing the pain and discomfort of the patient during injection, and is important for treating TNF- ⁇ -related diseases.
  • the present invention adds a stabilizer, a surfactant to the antibody composition, and further prepares the formulation of the antibody preparation as follows (1) or (2): (1) solvent water containing anti-TNF- ⁇ antibody The concentration is 20.0-130.0 mg/ml; the stabilizer contained includes one or two of 20.0-60.0 mg/ml polyol and 80.0-90.0 mg/ml sugar; the surfactant contained includes 0.5-2.0 mg/ Ml polysorbate 80; the formulation composition contained has a pH of 4.0 to 8.0, and the pH is adjusted by injecting carbon dioxide into the liquid component of the antibody composition at a pressure of 10-300 mmHg.
  • the surfactant comprises 3.0-10 mg/ml polysorbate 80;
  • the antibody composition has a pH of 4.0-6.0, and the pH is obtained by into the liquid component of the antibody composition 20-80mmHg pressure is pressed into the carbon dioxide to adjust.
  • the technical effect of the shelf life of the contained antibody component in the liquid state is 30 months, and the advantageous effect of the antibody composition being stored at room temperature for 2 weeks and the shelf life of 2 months at 2-8 ° C is 30 months;
  • the antibody composition exhibits excellent effects in terms of high temperature, light, antibacterial, and the like.
  • the inventor has further optimized the carbon dioxide pressure range through multiple experiments, and optimized the solute type and content of the antibody composition to realize the antibody combination.
  • the material is further improved in terms of shelf life, high temperature, light, and antibacterial.
  • Fig. 1 Results of room temperature stability test of the preparation of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406; wherein: Figure A is the size exclusion chromatography test result of each sample of Example 1.
  • Figure B is a graph showing the results of capillary electrophoresis test for each sample of Example 1;
  • Figure C is a graph showing the results of biological activity test of each sample of Example 1.
  • Figure 2 is a graph showing the results of a 15-day stability test under high temperature (40 ° C) conditions of two recombinant human anti-TNF- ⁇ monoclonal antibodies BAT1406;
  • Figure 3 is a graph showing the results of stability experiments of two recombinant preparations of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 under room temperature illumination (4000 lx);
  • Figure 4 is a diagram showing the results of a screening test for the mannitol concentration of the recombinant A anti-TNF- ⁇ monoclonal antibody BAT1406;
  • Figure 5A Effect of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 on experimental mouse tg197 in vivo arthritis score
  • Figure 5B Effect of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 on experimental histopathological score of experimental mouse tg197;
  • Figure 6 is a graph showing the blood concentration of the recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406A preparation when administered at low, medium and high doses;
  • Figure 7 is a graph showing the plasma concentration of the recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406A preparation and the buffer system-containing preparation.
  • the present invention provides an antibody composition against TNF- ⁇ , wherein the solvent comprises water, the solute comprises an anti-TNF- ⁇ antibody; the antibody composition has a pH of 4.0 to 8.0, and the pH is obtained by combining the antibodies
  • the liquid component of the substance is pressurized with carbon dioxide; further, the above composition further includes a surfactant and a stabilizer.
  • the antibody composition against TNF- ⁇ is preferably of two formulations, as follows:
  • Formula A solvent water, the concentration of the anti-TNF- ⁇ antibody contained is 20.0-130.0 mg/ml; the stabilizer contained includes one of 20.0-60.0 mg/ml polyol, 80.0-90.0 mg/ml sugar or Two kinds; the surfactant contained includes 0.5-2.0 mg/ml polysorbate 80; the antibody composition contained has a pH of 4.0-8.0, and the pH is obtained by into the liquid component of the antibody composition 10-300mmHg pressure is pressed into the carbon dioxide-regulated; the antibody composition prepared in the A formula is referred to as the A preparation, such as Table 1A below;
  • Formula B solvent water, the concentration of the anti-TNF- ⁇ antibody contained is 70.0-130.0 mg/ml; the stabilizer includes one of 3.0-15.0 mg/ml polyol, 5.0-8.0 mg/ml sodium chloride. Or both, the surfactant comprises 3.0-10 mg/ml polysorbate 80; the antibody composition has a pH of 4.0-6.0, and the pH is obtained by into the liquid component of the antibody composition 20-80mmHg pressure is pressed into the carbon dioxide to adjust.
  • An antibody composition prepared in Formula B is referred to as a B preparation, such as Table 1B below;
  • the carbon dioxide is charged after the preparation of the preparation.
  • a solvent system is prepared by dissolving the said mannitol (stabilizer) and polysorbate 80 (surfactant) components in about 90% by weight of the water for injection.
  • the mixed solution obtained in the step (3) is filled with carbon dioxide to adjust the pH, a final volume of water for injection is added, and then the preparation is sterilized by a filter membrane of a hydrophilic polyvinylidene fluoride having a pore size of 0.22 ⁇ m.
  • the filter membrane can filter the formulation into a sterile container.
  • the filter medium used is filter sterilized ammonia.
  • the formulation containing all of its ingredients was then filtered by filtration as described above, except that the formulation was filtered through two layers of sterile 0.22 ⁇ m membrane filters. After disinfection, the reagents are packaged for use in vials or pre-filled syringes, all of which are verified by microbial intrusion tests and meet the requirements of the Good Manufacturing Practice (2010 Revision).
  • weight and/or weight to volume ratios referred to herein may be converted to molar and/or molar concentrations using well known molecular weights of the ingredients.
  • the weights listed herein are for the volume.
  • the weight can be adjusted proportionally when different formulation volumes are required.
  • the 16L, 14L, 12L, 10L, 5L formulations each comprise 80%, 70%, 60%, 50%, 25% of the listed weight.
  • the preparation of the B preparation is the same as the preparation of the above A.
  • CE capillary electrophoresis
  • SEC size-exclusion chromatography
  • TNF- ⁇ -induced L929 cytotoxicity neutralization test The results of the assay are shown in Table 2B and 1.
  • Example 3 Study on factors affecting the pH of the preparation
  • BAT1406 Antibody A preparation (see Table 1A for specific formulation) 10L sample at a concentration of 100mg/mL, store in a pressure tank, charge different amounts of carbon dioxide, place at 4 ° C, test the pH of the preparation in the pressure tank when different carbon dioxide pressure is measured . The results of the measurements are shown in Table 3A.
  • the inventors have proved through many experiments that the preparation of the present invention can achieve better comfort when the carbon dioxide is pressurized at a pressure of 10-300 mmHg to adjust the pH to 4.0-8.0.
  • the results of the above examples show that when the carbon dioxide gas pressure ranges from 100 to 300 mmHg, the preparation pH is 4.76-5.84; the seal bottle of the preservation preparation A and the injection needle have good sealing property, and the charged carbon dioxide gas pressure is 220 mmHg. After storage for 15 days at room temperature, the pH of the preparation varied from 5.16 to 5.25, which basically met the experimental requirements.
  • the B formulation of BAT1406 (see Table 1B for specific formulation) has a gas pressure range of 40-60 mmHg, preferably 50 mmHg.
  • Example 4 Stability study under high temperature (40 ° C) conditions
  • BAT1406 antibody see Table 1A for specific formulation
  • preparation B see Table 1B for specific formulation
  • the samples were tested by IEC and the results of the measurements are shown in Figure 3.
  • the concentration of mannitol in the A preparation is from 60 mg/ml to 3 mg/ml, up to 18 mg/ml, and 15 (numbers 1-15, decreasing in concentration) are different.
  • the BAT1406 antibody A preparation (100 mg/ml) of mannitol concentration was different from Table 1A except that the A preparation mannitol concentration was partially the same as Table 1A.
  • the storage temperature was 40 ° C and the time period was 4 weeks. The samples were tested by SEC and IEC respectively, and the results are shown in Figure 4.
  • the mannitol concentration of the B formula was optimized, and it was found that mannitol can protect the BAT1406 antibody at 3-15 mg/ml, while the mannitol concentration is 10 mg/ml for the antibody. The protection is best.
  • Example 7 Microbial research
  • inoculating Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus to the A preparation (see Table 1A for specific formulation) and the B preparation (see Table 1B for specific formulation), respectively.
  • the amount was 100 cfu/ml, and the whole microbial growth in the inoculated preparation was measured by room temperature for 15 days after inoculation.
  • the evaluation indexes mainly included the number of microorganisms under the microscope and the change of the turbidity of the preparation, wherein the lack of turbidity indicates that there was no overall microbial growth. .
  • the microorganisms used in the examples include Escherichia coli (CICC-10003), Staphylococcus aureus (CICC-10306), Pseudomonas aeruginosa (WDCM-00024), and Bacillus cereus (CICC-10352).
  • test results are shown in Table 5: Storage at room temperature 20-25 ° C for 15 days, the A preparation, B preparation does not support microbial growth.
  • the insoluble particles in the preparation have a particle size of 1-50 ⁇ m, are invisible to the naked eye, can not be metabolized with blood flow, and may cause hard-to-find and potentially serious harm to the human body. Therefore, insoluble particle control is included in the injection quality standard.
  • A1 sample antibody partial concentration is 4mg/mL, the rest are the same as Table 1A;
  • A2 sample antibody partial concentration is 20mg/mL, the rest are the same as Table 1A;
  • A3 The sample composition is the same as Table 1A;
  • B1 sample antibody partial concentration is 4mg/mL, the rest is the same as Table 1B;
  • B2 sample antibody partial concentration is 20mg/mL, the rest are the same as Table 1B;
  • A3 The sample composition is the same as Table 1B;
  • the results show that the A preparation of the present invention has a shelf life of at least 30 months, and the B preparation has a shelf life of at least 24 months.
  • Example 10 Recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 protein sequence
  • BAT1406 The recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 was expressed in CHO cells by genetic engineering techniques and purified by a series of standard chromatographic steps.
  • BAT1406 is an IgG1 antibody with a molecular weight of approximately 148 kDa and consists of two IgG1 z, a heavy chains and two kappa light chains.
  • Each heavy chain contains 451 amino acids, a molecular weight of 49 kDa, and its heavy chain amino acid sequence is shown in SEQ ID No. 1.
  • Each light chain contains 214 amino acids with a molecular weight of 24 kDa and the light chain amino acid sequence is set forth in SEQ ID No. 2.
  • Example 11 Expression and purification of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406
  • a monoclonal antibody that specifically binds TNF- ⁇ is expressed in CHO cells according to the method of Wood et al., J Immunol. 145: 3011 (1990).
  • the expression vector containing the antibody gene was constructed by a conventional molecular biological method (Molecular Cloning) and expressed as a host cell of a CHO cell (ATCC CCL61).
  • the electroporated cells were immediately resuspended in 37 ° C pre-warmed CD CHO AGT TM medium, 100 ⁇ l per well was added to a 96-well plate, and 2-3 days later, an equal amount of screening medium (CDCHO AGT media + 50 uM methionine imino generation) was added. Sulfone (MSX)).
  • MSX Sulfone
  • the expression level of the antibody in the 96-well plate cell culture supernatant was measured.
  • the clones with higher expression levels were transferred from 96-well plates to 24-well plates. After the cells were grown to a certain amount, the cells were transferred to 6-well plates, and 2 ⁇ 10 5 cells were cultured in 5 ml of each well to measure the antibody production of the cells. And yield.
  • Example 12 Pharmacodynamic study of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406
  • Pharmacodynamic studies mainly carried out in vitro pharmacodynamic tests of recombinant human anti-TNF- ⁇ monoclonal antibodies and pharmacodynamic studies in in vivo animal models.
  • In vitro tests mainly carry out the following pharmacodynamic tests: binding ability to TNF, antibody specificity, competition for binding of TNF to receptor, inhibition of biological activity of TNF- ⁇ , and cytotoxicity experiments. .
  • the binding ability of recombinant human anti-TNF- ⁇ monoclonal antibody to TNF- ⁇ was compared by taking antibody specific binding as an example.
  • the specific experimental method is as follows: firstly dilute rhTNF- ⁇ , rhTNF- ⁇ or rmTNF- ⁇ into 50 ng/50 ⁇ l with PBS, and add diluted rhTNF- ⁇ , rhTNF- ⁇ or rmTNF- ⁇ to a micro 96-well microtiter plate. , 50 ⁇ l/well, overnight at 4 °C. The next day, it was blocked with PBS containing 3% BSA, 100 ⁇ l/well, and placed at 37 ° C for 2 hours.
  • the pharmacodynamic evaluation of animals mainly carried out acute animal models and chronic animal models.
  • the acute animal model is D-galactose-sensitive mouse and rhTNF- ⁇ -induced rabbit fever model.
  • a mouse model of intraperitoneal injection of 1 ⁇ g rhTNF- ⁇ +20mg D-galactosamine was used as a model to study the toxic effects of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 against rhTNF- ⁇ and D-galactosamine.
  • TNF- ⁇ monoclonal antibody injection has the effect of antagonizing the toxic effect of 1 ⁇ g rhTNF- ⁇ +20 mg D-galactosamine and improving the survival rate of mice.
  • the chronic animal model is a model of spontaneous polyarthritis in human soluble TNF transgenic mice (Tg197).
  • Tg197 stable expression of sTNF
  • the injection method used in this study was intraperitoneal injection.
  • the determination of the intraperitoneal injection protocol was based on the historical data of other anti-TNF- ⁇ monoclonal antibodies in the tg197 mouse test.
  • the transgenic mice were divided into 4 groups (G1). -G4): Each group contained 8 mice of different ages and genders.
  • the G1 group was injected with normal saline, and the G2-G4 group was injected with 3 mg/kg, 10 mg/kg, 30 mg/kg of BAT1406 antibody.
  • the experimental product was injected twice a week from the third week, starting from the third week, to the tenth week, before the formation of arthritis.
  • a control group of transgenic mice (4) was added, and the group was sacrificed before the first dose. All animals were sacrificed in the tenth week and serum and ankle joints were collected. Serum was stored at -80 °C for further analysis and the ankle joint was used for pathological evaluation.
  • the degree of arthritic lesions was assessed using a scoring system, and histopathological evaluation was performed using a pathology scoring system.
  • the experimental results show that the experimental product BAT1406 has a typical drug response in accordance with the pathological and in vivo inhibitory effects of Tg197 arthritis in the dose range of 3 mg/kg, 10 mg/kg, and 30 mg/kg.
  • the 10 mg/kg and 30 mg/kg doses of BAT1406 have a good inhibitory effect on the histopathological pathology and histopathology of the histological pathology. There is no statistical difference, and the scores obtained are lower than before treatment.
  • the BAT1406 antibody was shown to have a therapeutic effect on the tg197 arthritis model (see Figures 5A, 5B).
  • the purpose of this example is to evaluate the safety of the BAT1406 antibody preparation composition (A preparation) containing no buffer system in the BAT1406 preparation containing the buffer system as a control, and provide a reference for future clinical rational drug use.
  • the plasma concentration of the BAT1406 antibody preparation composition in a single dose of SD rats was determined by the BAT1406 antibody ELISA assay in SD rat plasma, and the high, medium and low doses of the BAT1406 antibody preparation composition were studied. , the relationship between exposure and dose of BAT1406 antibody in SD rats, and its pharmacokinetic characteristics; by comparing the kinetic characteristics of the same antibody dose BAT1406A preparation and BAT1406 buffer system preparation (pre-development) in SD rats, The bioequivalence of the two formulations of the buffer-free formulation of the present invention and the BAT1406 buffer-containing formulation (pre-developed) were evaluated.
  • the low, medium and high doses of BAT1406A preparation were 10mg/kg, 30mg/kg and 100mg/kg respectively (the dose here refers specifically to the dose according to the weight of SD rats), and the dose of BAT1406 containing buffer system is 30mg. /kg, 8 in each group, half male and half female.
  • the mode of administration is intraperitoneal injection.
  • the rats are cut at 1h, 4h, 24h, 48h, 72h, 96h, 7day, 9day, 11day, 13day, 15day, 22day, 29day after administration.
  • the blood sample was taken from the tail, and the collected blood sample was placed in an ice bath heparin anticoagulation centrifuge tube, centrifuged within 5 minutes, and the separated plasma was stored in a -70 ° C refrigerator for later use.
  • BAT1406 antibody content in plasma samples firstly coated with the recombinant rhTNF- ⁇ protein which specifically binds to the Fab segment of the BAT1406 antibody to capture the BAT1406 antibody in the biological sample, and then add the enzyme-labeled goat anti-human The antibody (IgG-HRP) was bound to the captured BAT1406 antibody, and finally the substrate was added for color development. After the reaction was terminated with a 2 mol/L sulfuric acid solution, the OD value of each well was read at 450 nm.
  • the absorbance of the BAT1406 antibody standard sample and the concentration of the standard sample were fitted to a standard curve by four parameters, and the concentration of the test sample 1406 antibody measured on the same plate was quantified using the obtained standard curve.
  • the mean of the pharmacokinetic parameters obtained from 8 rats represented the pharmacokinetic parameters of each dose group; the blood concentration of BAT1406 antibody at different doses and different preparation groups was statistically judged by independent sample t-test. The results are shown in Table 10 and Table 11.
  • the present embodiment provides a sample of the antibody composition, the raw material composition is shown in the Z1 sample and the Z2 sample in Table 12, and at the same time, a control (D-Z1 sample, D-Z2 sample) is also set for the Z1 sample and the Z2 sample, and The comfort performance of each sample was tested.
  • D-Z1 sample, D-Z2 sample is also set for the Z1 sample and the Z2 sample, and The comfort performance of each sample was tested.
  • Pain test refer to birtha Hansen et al. Pain Perception after Subcutaneous Injections of Media Containing Different Buffers. Basic & Clinical Pharmacology & Toxicology. 2006, 98, 218-221. The test results are shown in Table 13.
  • This example provides a sample of an antibody preparation obtained by formula A.
  • the composition of the raw materials is shown in Table 14, and the performance of each sample was tested.
  • the test results are shown in Table 15.
  • This example provides a sample of the antibody preparation sample composition obtained by the B formula.
  • the composition of the raw materials is shown in Table 16, and the performance of each sample was tested.
  • the test results are shown in Table 17.
  • This comparative example provides the following four comparative samples, wherein: D1 sample and D2 sample are comparative samples of the A1 sample in Example 15, and D3 sample and D4 sample are comparative samples of the B1 sample in Example 16, the comparative sample is composed as follows Table 18 shows the results of the detection of each sample as shown in Table 19 below.

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Abstract

La présente invention concerne une composition d'anticorps anti-TNF-α. Le solvant de la composition comprend l'eau, et le soluté de la composition comprend un anticorps anti-TNF-α. La valeur de pH de la composition est de 4,0 à 8,0, et la valeur de pH est ajustée par introduction de dioxyde de carbone dans le solvant et le mélange de soluté de la composition. Comparativement à l'état de la technique, la composition d'anticorps selon la présente invention ne contient pas de système tampon commun, et du dioxyde de carbone est introduit pour remplacer ledit système tampon et maintenir à l'état stable la valeur de pH d'une préparation; ainsi, les douleurs et l'inconfort du patient pendant l'injection sont réduits, et la composition d'anticorps est d'une signification majeure pour le traitement des maladies associées au TNF-α.
PCT/CN2018/097445 2017-07-31 2018-07-27 COMPOSITION D'ANTICORPS ANTI-TNF-α, ET SON APPLICATION WO2019024783A1 (fr)

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