WO2019154397A1 - 一种靶向治疗TNF-α相关疾病的人抗体制剂 - Google Patents

一种靶向治疗TNF-α相关疾病的人抗体制剂 Download PDF

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WO2019154397A1
WO2019154397A1 PCT/CN2019/074715 CN2019074715W WO2019154397A1 WO 2019154397 A1 WO2019154397 A1 WO 2019154397A1 CN 2019074715 W CN2019074715 W CN 2019074715W WO 2019154397 A1 WO2019154397 A1 WO 2019154397A1
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preparation
tnf
antibody
hcl
buffer
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French (fr)
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吴用
林键
裴树军
王盛武
莫文俊
李胜峰
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百奥泰生物制药股份有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K47/16—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
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    • 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
    • A61K47/26—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
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A—HUMAN NECESSITIES
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    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to an antibody preparation targeted to treat TNF- ⁇ related diseases, which is mainly a liquid aqueous pharmaceutical preparation.
  • RA Rheumatoid arthritis
  • TNF- ⁇ tumor necrosis factor- ⁇
  • interleukins such as IL-1, IL-6, IL-8, etc.
  • the main mechanisms of action of these biological agents are: 1) monoclonal antibodies directed against cytokines or their receptors; (2) soluble receptor antagonists, ie cytokine surface receptors that do not include transmembrane components and intracellular functional regions.
  • the receptor antagonist binds to free cytokines and inhibits 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 integrated structures such as Fc receptors of IgG or polyethylene glycol (PEG); (3) receptor antagonistic proteins, none A biologically active protein that 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 ⁇ Tumor Necrosis Factor ⁇ , tumor necrosis factor- ⁇
  • TNF ⁇ Tumor Necrosis Factor ⁇ , tumor necrosis factor- ⁇
  • the levels of TNF- ⁇ and TNFR in RA were significantly elevated in serum, synovium, and synovial fluid, especially in patients with severe disease and active 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.
  • ESR erythrocyte sedimentation rate
  • TNF can act on a variety of cells, such as promoting macrophage secretion of inflammatory cytokines and chemokines, and promoting inflammation.
  • the main functions of TNF- ⁇ are: (1) Inducing endothelial cells to express adhesion molecules and vascular endothelial growth factor (VEGF), promoting adhesion and penetration of leukocytes and vascular endothelium, leading to local inflammatory reaction and vascular VEGF production; (2) acting on Hepatocytes produce C-reactive protein (CRP); (3) TNF- ⁇ can act on osteoclasts, synoviocytes and chondrocytes in rheumatoid arthritis, resulting in activation of these cells, producing metalloproteinases and collagenases.
  • VEGF vascular endothelial growth factor
  • CRP C-reactive protein
  • TNF- ⁇ can also promote synovial cells, macrophages, fibroblasts and Chondrocytes produce IL-1, IL-8 and TNF- ⁇ themselves and aggravate tissue damage. Therefore, inhibition of TNF- ⁇ is important for controlling the condition of RA and improving prognosis [3] .
  • TNF- ⁇ inhibitors There are currently three TNF- ⁇ inhibitors approved by the US FDA: soluble receptor antagonist-Etanercept, human-mouse chimeric antibody-Infliximab and humanized mAb .
  • Etanercept is a recombinant human type II tumor necrosis factor receptor-antibody fusion protein, which is composed of a type II TNF receptor (p75) and a Fc part of IgG1;
  • Infliximab is a specific IgG1 monoclonal antibody against TNF (by human Ig)
  • a humanized mAb is also an IgG1 monoclonal antibody specific for TNF (both stable and variable regions of Ig are of human origin).
  • the main adverse reactions of TNF inhibitors are injection site reactions, infections, tumors, lymphoproliferative disorders, neurodegenerative lesions, and lupus-like syndromes [4-5]
  • the human antibody BAT1406 preparation previously developed by the applicant for the treatment of TNF- ⁇ -related diseases has an antibody concentration of 20-80 mg/mL (CN103446583A), which is difficult to exceed the upper limit of 80 mg/mL. Despite such a need, there is currently no satisfactory formulation for this antibody that supports high concentrations of antibodies.
  • Another object of the present invention is to provide a higher concentration antibody preparation.
  • Another object of the present invention is to provide a human antibody preparation for treating a TNF- ⁇ -related disease with higher stability.
  • the invention provides an antibody preparation comprising an aqueous pharmaceutical preparation comprising a recombinant human anti-TNF- ⁇ monoclonal antibody, a buffer system, a stabilizer, a surfactant.
  • an antibody preparation of the present invention contains the following components:
  • the antibody preparation has a pH of 4.0 to 7.0.
  • the recombinant human anti-TNF- ⁇ monoclonal antibody is expressed in CHO cells by genetic engineering and purified by a series of standard chromatographic steps. After preparation of the antibody, a pharmaceutical preparation is prepared.
  • the recombinant human anti-TNF- ⁇ monoclonal antibody has the heavy chain of SEQ ID NO. 1 and the light chain of SEQ ID NO. 2 (named BAT1406).
  • the TNF- ⁇ monoclonal antibody or antigen-binding portion thereof is capable of binding to human TNF- ⁇ .
  • the antibody has a concentration of 40 to 120 mg/ml, more preferably 50 to 100 mg/ml.
  • the buffering agent is selected from one or a combination of succinic acid, citric acid, His-HCl, acetic acid, phosphate; preferably, the buffer is selected from the group consisting of His-HCl, HAc, and phosphoric acid. Combination of salt and HAc, combination of His-HCl and HAc; more preferably a combination of His-HCl and HAc.
  • each of the buffers has a concentration of 5 to 15 mM; more preferably 5 to 10 mM. More preferably, when the buffering agent is selected from the combination of the two, the molar ratio of the former to the latter in the system is from 1:2 to 2:1; more preferably 1:1.
  • the stabilizer is 0.5% to 5.0% Arg-HCl and 0.01% to 2% Met; or is selected from 3.0% to 6.0% sorbitol; or alternatively selected from 7% to 10% trehalose.
  • the surfactant is selected from the group consisting of Tween 80, Tween 20 or Poloxamer 188.
  • the surfactants selected for this formulation are nonionic surfactants which enhance the stability of the formulation, especially under shaking and repeated freeze-thaw conditions.
  • the inventors have found that in the formulations of the present invention, different types of nonionic surfactants have little effect on the improvement of stability.
  • Tween 80, Tween 20, and Poloxamer 188 are all nonionic surfactants. They are used in biomimetic preparations in an amount of 0.001% to 0.1%. The amount used is very low, and the surface activity is very small. The agent can meet the stability requirements of the sample.
  • the surfactant is an amphiphilic molecule having a hydrophilic group and a lipophilic group
  • the two phases are regarded as components of the phase by the affinity of different portions of the molecule for the two phases, respectively.
  • the surface of the two phases is equivalent to transfer into the interior of the molecule, thereby reducing the surface tension. Since both phases regard it as a component of the phase, it is equivalent to the absence of an interface between the two phases and the surfactant molecules. In this way, the interface between the two phases is partially eliminated, thus reducing the surface tension. And the surface free energy, in order to achieve the purpose of stabilizing the monoclonal antibody.
  • the antibody preparation of the present invention contains the following components:
  • the pH of the preparation is 5.0 to 5.6, preferably, the pH is 5.2 to 5.6;
  • the pH of the preparation is 5.0 to 5.6, preferably, the pH is 5.0 to 5.4;
  • the pH of the preparation is from 5.0 to 5.6, preferably, the pH is from 5.0 to 5.4.
  • the antibody preparation of the invention comprises the following components:
  • the pH of the preparation is 5.0 to 5.6, preferably, the pH is 5.2 to 5.6;
  • the pH of the preparation is 5.0 to 5.6, preferably, the pH is 5.0 to 5.4;
  • an effective amount of anti-TNF- ⁇ human antibody of 80 to 120 mg/ml; preferably 85 to 120 mg/ml; more preferably 90 to 110 mg/ml.
  • the formulation has a pH of from 5.0 to 5.6, preferably a pH of from 5.0 to 5.4.
  • the formulation contains the following components:
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.4 ⁇ 0.2;
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.2 ⁇ 0.2;
  • the formulation has a pH of 5.3 ⁇ 0.3, preferably a pH of 5.2 ⁇ 0.2.
  • the formulation contains the following components:
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.4 ⁇ 0.2;
  • the formulation contains the following components:
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.4 ⁇ 0.2;
  • the formulation contains the following components:
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.2 ⁇ 0.2;
  • the formulation contains the following components:
  • the pH of the preparation is 5.3 ⁇ 0.3, preferably, the pH is 5.2 ⁇ 0.2;
  • the formulation contains the following components:
  • the formulation has a pH of 5.3 ⁇ 0.3, preferably a pH of 5.2 ⁇ 0.2.
  • the formulation contains the following components:
  • the formulation has a pH of 5.3 ⁇ 0.3, preferably a pH of 5.2 ⁇ 0.2.
  • the formulation of the invention also contains NaOH for pH adjustment.
  • the formulation of the present invention has an osmotic pressure of from 150 to 400 mOsm/kg; as a preferred embodiment, the osmotic pressure is from 260 to 340 mOsm/kg.
  • the present invention provides a method of preparing the above antibody preparation, comprising the steps of:
  • the formulation Prior to filling the main container, the formulation was sterile filtered using a 0.2 micron filter in a clean bench. The filling is also performed aseptically.
  • the aqueous liquid antibody preparation of the present invention is suitable for subcutaneous injection.
  • the concentration of the recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 can be as high as 120 mg/ml, for example, the antibody can be stably present in the concentration range of 20 to 120 mg/ml, and the BAT1406 antibody preparation in CN103446583A
  • the antibody concentration is only up to 80 mg/ml (CN103446583A), and the present invention increases the upper limit by 50%.
  • the antibody preparation of the present invention has high stability and can prolong the storage period of BAT1406 in an aqueous preparation; in particular, the preparation is stable at room temperature at least one Month; stable for at least 36 months at 2-8 ° C; maintain stability after at least 5 cycles of freeze-thaw. More specifically, BAT1406 is stable for one month at room temperature in an aqueous formulation; 36 months at 2-8 ° C; and after 25 cycles of freeze-thaw, the EC50 is below 22 ng/ml, more preferably below 21 ng/ml.
  • the recombinant human anti-TNF- ⁇ monoclonal antibody preparation developed by the invention has high concentration of antibody protein, is more stable, and is more resistant to aggregation reaction caused by high temperature change, so the antibody preparation of the invention can be used for preserving targeted therapy TNF.
  • - ⁇ -related human antibodies enhance the stability of TNF- ⁇ antagonist antibody drugs including recombinant human anti-TNF- ⁇ monoclonal antibodies, and prolong their preservation period in aqueous preparations, for targeted treatment of TNF- ⁇ related diseases Significance.
  • Figure 8 Number of insoluble particles ( ⁇ 10 ⁇ m) after dilution of different concentrations of surfactant samples (pcs/mL)
  • Figure 18a Subcutaneous injection of the recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 by subcutaneous injection of A1, B1, C1 in vivo
  • FIG. 18b Subcutaneous injection of the recombinant A3, B2, and C2 recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 in vivo
  • the "%" of the component refers to the mass volume (w/v) percentage, for example, a component content of 6%, that is, 100 ml of the formulation system. Contains 6g of this component.
  • stable refers to a liquid formulation comprising an antibody (including antibody fragments thereof) in which the antibody (including antibody fragments thereof) does not occur under the given conditions of manufacture, preparation, transport and/or storage. Aggregation, degradation or fragmentation occurs with little or no effect.
  • a “stable” formulation retains biological activity under the given conditions of manufacture, preparation, transport and/or storage. The stability of the antibody (including antibody fragments thereof) can be assessed by the degree of aggregation, degradation or fragmentation of the formulation as measured by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS, and the like.
  • the buffer is configured by a buffer to maintain the pH of the solution relatively stable to a certain extent.
  • Common buffers such as salts of organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; Tris, tromethamine hydrochloride, or phosphate; A buffer such as glycine, histidine or the like.
  • Common buffer systems such as: phthalate-hydrochloride buffer, disodium hydrogen phosphate-citrate buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, Acetic acid-sodium acetate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, phosphate buffer (disodium hydrogen phosphate-sodium dihydrogen phosphate buffer), barbital sodium-hydrochloric acid buffer, Tris-HCl buffer Boric acid-borax buffer, glycine-sodium hydroxide buffer, glycine-hydrochloric acid buffer, borax-sodium hydroxide buffer, sodium carbonate-sodium bicarbonate buffer, and the like.
  • the above buffer system can be applied to the antibody preparation of the present invention to maintain the pH of the system.
  • the choice of buffer will still affect the stability of the antibody preparation of the present invention more critically.
  • the choice of buffer system appears to be more important.
  • the invention has experimentally screened a series of suitable buffer systems: succinic acid buffer, citrate buffer, histidine buffer, acetate buffer, phosphate buffer + citrate buffer, phosphate buffer Liquid + Acetate Buffer, Histidine Buffer + Succinic Acid Buffer, Histidine Buffer + Citric Acid Buffer, Histidine Buffer + Acetate Buffer, Succinic Acid Buffer + Citric Acid Buffer, Succinic Acid Buffer + acetate buffer.
  • the most preferred buffer system is histidine buffer + acetate buffer. It was found that the histidine component in the buffer can improve the stability of SEC and IEC of the sample, and the acetic acid component does not affect the stability of the sample. On the basis of, it can provide a stable pH buffer range for the buffer system, so histidine buffer + acetate buffer is preferred.
  • Histidine is a basic amino acid.
  • a histidine buffer is prepared, as a non-limiting example, after the histidine is dissolved in water, the pH is adjusted with hydrochloric acid to form a histidine buffer; The added hydrochloric acid reacts with a part of histidine to form histidine hydrochloride, and therefore, histidine and histidine hydrochloride are present in the buffer.
  • Histidine buffers of different pH affect the ratio of histidine to histidine hydrochloride, and the molar concentration of histidine is the sum of histidine and histidine hydrochloride. . Therefore, in the present invention, the histidine buffer is also expressed as the abbreviation "His-HCl".
  • the stabilizer in the preparation has an effect of regulating the osmotic pressure, so the stabilizer can also be called an osmotic pressure regulator.
  • the preparation provided by the present invention is a liquid aqueous medicine having a pH of 4.0 to 7.0, and the preparation contains an antibody having a concentration of 20 to 120 mg/ml, and has an enhanced TNF- ⁇ such as a recombinant human anti-TNF- ⁇ monoclonal antibody.
  • the antibody concentration of the preparation may be less than 20 mg/ml. However, depending on the amount of administration, an increase in volume is required. In order to reduce the administration volume and the number of administrations, the pain of the patient is reduced, and the risk of infection due to the number of administrations is controlled. Therefore, increasing the concentration is preferred.
  • the preparation of the present invention comprises the following components: an antibody capable of binding human hTNF- ⁇ with high affinity, low dissociation rate, high neutralizing ability; buffer, including His-HCl, or HAc, or a combination of phosphate and HAc, or His a combination of HCl and HAc; an osmotic pressure adjusting agent comprising one or both of sorbitol, trehalose, arginine and methionine; a surfactant comprising Tween 80; a pH-adjusting sodium hydroxide and Water for Injection.
  • the concentration of the antibody in the liquid aqueous pharmaceutical formulation is from about 40 to 120 mg/ml, preferably from 45 to 110 mg/ml.
  • the antibody is a recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406, and BAT1406 is an adalimumab biosimilar.
  • the present invention provides aqueous antibody preparations of different buffer systems, including succinic acid buffer, citrate buffer, histidine buffer, acetate buffer, phosphate buffer + citrate buffer, phosphate buffer + acetate buffer.
  • succinic acid buffer citrate buffer
  • histidine buffer acetate buffer
  • phosphate buffer + citrate buffer citrate buffer
  • phosphate buffer + acetate buffer Liquid, histidine buffer + succinic acid buffer, histidine buffer + citrate buffer, histidine buffer + acetate buffer, succinate buffer + citrate buffer, succinate buffer + acetic acid
  • the buffer has a pH of about 3.0 to 6.0.
  • the present invention provides a liquid aqueous antibody preparation comprising a stabilizer (polyol) to adjust the osmotic pressure of the liquid system to stabilize the antibody.
  • the stabilizer is added to the antibody in an amount that can vary depending on the isotonicity of the formulation desired.
  • arginine hydrochloride and methionine are used as the osmotic pressure adjusting agent in the preparation, and in a preferred embodiment of the present invention, the stabilizer is selected from 3.0.
  • sorbitol preferably sorbitol at a concentration of 4.2%; or from 3.5% to 5.5% mannitol, preferably mannitol at a concentration of 4.5%; or 0.9% sodium chloride; or 1.0% to 5.0% hydrazine
  • concentration of the proline, preferably valine is 2.5%; or 3.5% to 9% sucrose, preferably sucrose is 6%; or 7.0% to 10% trehalose, preferably trehalose is 8.4%; or 0.5% ⁇ 5.0% arginine hydrochloride, preferably arginine hydrochloride, has a concentration of 3.1%; or 0.1% to 2% methionine.
  • the formulation has an osmotic pressure in the range of from 150 to 400 mOsm/kg.
  • the present invention provides liquid aqueous antibody preparations comprising a buffer system.
  • the buffer system includes a histidine buffer, an acetate buffer or a phosphate buffer.
  • the buffer system is a histidine buffer and an acetate buffer, and the concentration of the histidine buffer and the acetate buffer is in the range of 5 to 15 mM, preferably 5 to 10 mM. More preferably, the concentration is 5 mM.
  • the buffer system is a histidine buffer, an acetate buffer, and a phosphate buffer.
  • One of the buffers in this protocol is a histidine buffer at a concentration ranging from 5 to 15 mM, preferably at a concentration of 5 mM.
  • Another buffer in this protocol is an acetate buffer at a concentration ranging from 10 to 25 mM, preferably at a concentration of 17 mM.
  • Another buffer in this protocol is a phosphate buffer at a concentration ranging from 5 to 15 mM, preferably at a concentration of 10 mM.
  • the buffer system is a histidine buffer and an acetate buffer.
  • One of the buffers in this protocol is a histidine buffer at a concentration ranging from 15 to 25 mM, preferably at a concentration of 20 mM.
  • Another buffer in this protocol is an acetate buffer at a concentration ranging from 10 to 25 mM, preferably at a concentration of 17 mM.
  • the buffer system is acetate buffer and phosphate buffer.
  • One of the buffers in this protocol is an acetate buffer at a concentration ranging from 10 to 25 mM, preferably at a concentration of 17 mM.
  • Another buffer in this protocol is a phosphate buffer at a concentration ranging from 5 to 15 mM, preferably at a concentration of 10 mM.
  • the present invention provides liquid aqueous antibody preparations which include a surfactant.
  • Typical surfactants include nonionic surfactants such as Tween 80.
  • Surfactants can reduce aggregation of the prepared antibodies and/or reduce the formation of particles in the formulation and/or reduce adsorption.
  • the formulation uses Tween 80 as a surfactant.
  • the formulation comprises from about 0.01% to about 0.2% Tween 80, more preferably from 0.01% to 0.1%, and more preferably, about 0.1% Tween 80 is present in the formulation of the invention.
  • the preparation buffer system comprises a histidine buffer and an acetate buffer.
  • Stabilizers for the formulation include sorbitol, or trehalose, or a combination of arginine hydrochloride and methionine.
  • Surfactants for the formulation include Tween 80.
  • the pH of the formulation ranges between 4.0 and 7.0.
  • the formulation has an osmotic pressure in the range of from 150 to 400 mOsm/kg.
  • the buffer system is a histidine buffer and acetic acid, and the concentration ranges from 5 to 15 mM, preferably from 5 to 10 mM, more preferably at a concentration of 5 mM, and the pH ranges from 4.0 to 7.0.
  • the pH is from 5.0 to 5.6, more preferably from 5.2 to 5.6.
  • the concentration of arginine hydrochloride and methionine as stabilizers is 0.5% to 5.0% and 0.01% to 2%, respectively, and the preferred concentrations are 3.1% and 0.1%, respectively.
  • What is used as a surfactant is Tween 80, which has a concentration ranging from 0.01% to 0.2%, preferably 0.1%.
  • the formulation has an osmotic pressure in the range of 150 to 400 mOsm/Kg and an optimized osmotic pressure of 260 to 340 mOsm/kg.
  • the buffer system is a histidine buffer and acetic acid, and the concentration ranges from 5 to 15 mM, preferably from 5 to 10 mM, more preferably at a concentration of 5 mM, and the pH ranges from 4.0 to 7.0, preferably pH is in the range of 5.0 to 5.6, and more preferably in the range of 5.0 to 5.4.
  • the sorbitol as a stabilizer has a concentration ranging from 3% to 6%, preferably at a concentration of 4.2%.
  • Tween 80 Used as a surfactant is Tween 80, which has a concentration ranging from 0.01% to 0.5%, preferably 0.1%.
  • the formulation has an osmotic pressure in the range of from 150 to 400 mOsm/kg, preferably an osmotic pressure of from 260 to 340 mOsm/kg.
  • the buffer system is a histidine buffer and acetic acid, and the concentration ranges from 5 to 15 mM, preferably from 5 to 10 mM, more preferably at a concentration of 5 mM, and the pH ranges from 4.0 to 7.0, preferably pH is 5.0 to 5.6, and more preferably pH is 5.0 to 5.4.
  • trehalose has a concentration ranging from 7% to 10%, preferably at a concentration of 8.4%.
  • What is used as a surfactant is Tween 80, which has a concentration ranging from 0.01% to 0.2%, preferably 0.1%.
  • the formulation has an osmotic pressure of from 150 to 400 mOsm/kg, preferably an osmotic pressure of from 260 to 340 mOsm/kg.
  • the present invention provides liquid aqueous pharmaceutical preparations, including antibodies suitable for therapeutic use, which are convenient for administration and which contain high protein concentrations, primarily for the treatment of conditions caused by TNF-[alpha].
  • the pharmaceutical formulation has the effect of enhancing stability.
  • the formulations of the invention are stable after at least 5 freeze-thaw cycles.
  • the formulation of the invention remains stable after being placed at room temperature for one month.
  • the formulations of the invention are stable for at least 36 months at 2-8 °C.
  • the antibody is directed against human TNF-[alpha].
  • the antibody is recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406.
  • the liquid aqueous pharmaceutical preparation comprises an antibody or antigen binding portion thereof, which is a recombinant antibody or antigen binding portion thereof, which inhibits human TNF- ⁇ -induced ELAM-1 in human veins Expression on endothelial cells.
  • the formulation comprises recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 having two heavy chains of SEQ ID NO. 1 and two light chains of SEQ ID NO. .
  • the invention provides an aqueous pharmaceutical preparation comprising an effective amount of an antibody component, the buffer system is a histidine buffer and an acetate buffer, and the osmotic pressure stabilizer is arginine hydrochloride and methionine, or sorbitol, Or trehalose, surfactant Tween 80 and water for injection, pH is about 4.0-7.0.
  • the formulation is suitable for subcutaneous injection.
  • the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 100 mg/ml; in one embodiment of the invention, the antibody is in the liquid aqueous pharmaceutical formulation The concentration is about 80 mg/ml; in another embodiment of the invention, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 50 mg/ml.
  • the formulation is a solution containing the ingredients shown in Tables 1a-1c; preferably, the formulation is contained in a prefilled syringe.
  • the preparation may comprise a component of 40 mg effective amount of anti-hTNF- ⁇ antibody; 3.1% arginine hydrochloride with 0.1% methionine, 0.1% Tween 80, 5 mM histidine hydrochloride and 5 mM acetic acid. Sodium hydroxide and water for injection for pH adjustment (see Table 1a).
  • the preparation may further comprise an effective amount of 40 mg of anti-hTNF- ⁇ antibody; 4.2% sorbitol, 0.1% Tween 80, 5 mM histidine hydrochloride and 5 mM acetic acid, sodium hydroxide and water for injection for pH adjustment. (See Table 1b).
  • the preparation may further comprise an effective amount of 40 mg of anti-hTNF- ⁇ antibody; 8.4% trehalose, 0.1% Tween 80, 5 mM histidine hydrochloride and 5 mM acetic acid, sodium hydroxide and water for injection for pH adjustment. (See Table 1c).
  • Table 1a List of A1 formulation components of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 (antibody concentration 100 mg/mL)
  • Table 1b List of B1 formulation components of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 (antibody concentration 100 mg/mL)
  • Antibody BAT1406 40mg Active substance Sorbitol 4.2% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 400ul Solvent
  • Table 1c List of C1 formulation components of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 (antibody concentration 100 mg/mL)
  • Active substance Antibody BAT1406 40mg Active substance Trehalose 8.4% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 400ul Solvent
  • the formulation contains a solution of the ingredients shown in Tables 1d-1f; preferably, the formulation is contained in a prefilled syringe.
  • Active substance Antibody BAT1406 40mg Active substance Arginine hydrochloride 3.1% Osmotic pressure regulator Methionine 0.1% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 500ul Solvent
  • Antibody BAT1406 40mg Active substance Sorbitol 4.2% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 500ul Solvent
  • Active substance Antibody BAT1406 40mg Active substance Trehalose 8.4% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 500ul Solvent
  • the formulation contains a solution of the ingredients shown in Tables 1g-1i; preferably, the formulation is contained in a prefilled syringe.
  • Active substance Antibody BAT1406 40mg Active substance Arginine hydrochloride 3.1% Osmotic pressure regulator Methionine 0.1% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 800ul Solvent
  • Antibody BAT1406 40mg Active substance Sorbitol 4.2% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 800ul Solvent
  • Active substance Antibody BAT1406 40mg Active substance Trehalose 8.4% Osmotic pressure regulator Histidine hydrochloride 5mM Buffer acetic acid 5mM Buffer Tween80 0.1% Surfactant Sodium hydroxide Adjust pH to 5.3 ⁇ 0.3 pH regulator Water for Injection Added to 800ul Solvent
  • the present invention provides a liquid aqueous drug.
  • the preparation may comprise an antibody concentration of 40 to 120 mg/ml, 5 to 15 mM histidine buffer, and 5 to 15 mM acetate buffer. 0.5% to 5.0% arginine hydrochloride, 0.01% to 2% methionine, 0.01% to 0.2% Tween 80 and water for injection, pH 4.0 to 7.0.
  • a preferred embodiment comprises an antibody concentration of 45-110 mg/ml, 5 mM histidine buffer, 5 mM acetate buffer, 3.1% arginine hydrochloride, 0.1% methionine, 0.1% Tween 80 and water for injection, pH 5.3. ⁇ 0.3, more preferably pH 5.4 ⁇ 0.2, pH-adjusted sodium hydroxide and water for injection (hereinafter referred to as Formula A).
  • the preparation may comprise a component concentration of 40 to 120 mg/ml of antibody, 5 to 15 mM histidine buffer, 5 to 15 mM acetate buffer, 3.0% to 6.0% sorbitol, and 0.01%. 0.2% Tween 80 and water for injection, pH 4.0-7.0.
  • a preferred embodiment comprises an antibody concentration of 45 to 110 mg/ml, 5 mM histidine buffer, 5 mM acetate buffer, 4.2% sorbitol, 0.1% Tween 80 and water for injection, pH 5.3 ⁇ 0.3, more preferably pH 5.2 ⁇ 0.2. , pH-adjusting sodium hydroxide and water for injection (hereinafter referred to as B preparation).
  • the preparation may comprise a component concentration of 40 to 120 mg/ml, 5 to 15 mM histidine buffer, 5 to 15 mM acetate buffer, 7% to 10% trehalose, and 0.01% to ⁇ . 0.2% Tween 80 and water for injection, pH 4.0-7.0.
  • a preferred embodiment comprises an antibody concentration of 45 to 110 mg/ml, 5 mM histidine buffer, 5 mM acetate buffer, 8.4% trehalose, 0.1% Tween 80 and water for injection, pH 5.3 ⁇ 0.3, more preferably pH 5.2 ⁇ 0.2. , pH-adjusting sodium hydroxide and water for injection (hereinafter referred to as C preparation).
  • the formulations of the invention are stable after at least 5 freeze-thaw cycles.
  • the aqueous pharmaceutical preparation can be kept stable at 2-8 ° C for 36 months.
  • the liquid aqueous pharmaceutical preparations are primarily used to treat conditions caused by TNF-[alpha].
  • the formulation is suitable for subcutaneous injection.
  • the liquid aqueous pharmaceutical preparation can enhance the stability of a drug containing a TNF- ⁇ antagonist antibody such as a recombinant human anti-TNF- ⁇ monoclonal antibody, and prolong its preservation period in an aqueous preparation.
  • Histidine hydrochloride histidine buffer, histidine, and His-HCl are understood herein to have the same meaning, that is, a buffer configured using histidine and hydrochloric acid.
  • the preparation method of 11 groups of buffer systems containing antibodies is as follows:
  • sample components were: 10 mM buffer plus BAT1406 antibody at an antibody concentration of 50 mg/mL. Since this experiment is a screening buffer, no other ingredients have been added yet.
  • Preparation method Weigh 10 mM buffer, dissolve in water, adjust the pH to 5.2 with sodium hydroxide, and make up to volume. Finally, 11 sets of samples were prepared by ultrafiltration. The ultrafiltration method is described above.
  • the stability of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 was studied on the 0th, 5th, 10th, and 15th day in a certain pH range, and the SEC main peak, polymer and fragment were analyzed respectively; IEC main peak And acid region analysis; CE (NR) main peak and fragment analysis; DSC detection.
  • the capillary electrophoresis apparatus uses Beckman's PA800plus. After the sample is injected, the chromatogram is recorded, the data is integrated, and the percentage of the monomer peak is calculated by the area normalization method.
  • No. 1 to 11 in Table 1 correspond to the serial numbers A160923-1 to A160923-11 in the figure.
  • the preparation method of the above preparation sample is:
  • buffers 1 to 4 (1) Prepare a buffer. Taking 1L as an example, the combination preparation methods of buffers 1 to 4 are: 1.05g histidine hydrochloride, 0.834g acetic acid, dissolved in water, adjusted to pH 5.2 with sodium hydroxide, constant volume To 1L; weigh 2.10g histidine hydrochloride, 2.836g acetic acid, 1.38g disodium hydrogen phosphate, dissolved in water, adjust the pH to 5.2 with sodium hydroxide, dilute to 1L; weigh 4.19g histidine Hydrochloride, 2.836g acetic acid, dissolved in water, adjusted to pH 5.2 with sodium hydroxide, dilute to 1L; weigh 2.836g acetic acid, 1.38g disodium hydrogen phosphate, dissolved in water, adjust the pH with sodium hydroxide 5.2, make up to 1L.
  • the formulation Prior to filling the main container, the formulation was sterile filtered using a 0.2 micron filter in a clean bench. The filling is also performed aseptically.
  • sample names A161224-1/A161224-2/A161224-3/A161224-4 in Table 6 correspond to the serial numbers 1/2/3/4 in Table 5, respectively.
  • Stabilizer screening test (high temperature 40 ° C): At the high temperature of 40 ° C, 11 kinds of stabilizers (see Table 7) were analyzed on the 0th, 5th, 10th, and 15th days of SEC main peak, IEC main peak and acid region. The detection method is the same as in the first embodiment.
  • Stabilizer screening test (lighting 4000lx): SEC main peak, IEC main peak and acid region, CE, on the 0th, 5th, 10th and 15th day of the above samples with different stabilizers (see Table 7) under illumination 4000lx, CE - Analysis of SDS (NR). See the examples for the detection method.
  • This test was carried out to compare the protective effects of the three surfactants on the antibody protein to determine the surfactant concentration and to effectively protect the stability of the antibody.
  • a protein solution containing different concentrations of surfactant is prepared.
  • the specific formula is shown in Table 9.
  • the antibody protein concentration in the solution was 100 mg/mL, 80 mg/mL, 50 mg/mL, the protein (BAT1406) contained no surfactant, and the other components (5 mM HAc, 5 mM His-HCl, 3.1% Arg-HCl, 0.1% Met) , pH 5.3) are contained.
  • the antibody protein (BAT1406) concentration is 100mg/mL, 80mg/mL, 50mg/mL, respectively, sub-package, high temperature test, high temperature 1 week (1w) and high temperature 2 weeks (2w)
  • the samples were tested by SEC-HPLC and compared with samples without high temperature (0D).
  • Tables 10a-c The results are shown in Tables 10a-c:
  • the antibody protein (BAT1406) concentration is 100mg/mL, 80mg/mL, 50mg/mL, respectively, according to the ratio (100 ⁇ L sample: 20mL 0.9% sodium chloride solution) In 0.9% NaCl solution (normal saline), the traits were observed after slight and thorough mixing, and the insoluble particles were detected by the protein solution placed for 2 hours.
  • the method for detecting insoluble particles is: in accordance with the "People's Republic of China Pharmacopoeia 2015 Edition Part IV" General Rule 0903: Insoluble Particle Inspection Method. After the instrument has been cleaned to the pass, carefully mix the contents of the four test articles in the clean bench, place them in the sampling cup, let stand for 2 minutes, remove the air bubbles, and place them on the sampler. Turn on the agitation to mix the solution (to avoid air bubbles), and measure it 4 times according to the law. The injection volume is 4.0ml each time. The first measurement data is discarded, and the average value of the subsequent three measurement data is taken as the measurement result, and the number of particles contained in each container is calculated based on the sampling volume and the volume of the labeling device of each container.
  • the particle mean data in Table 12 is plotted to obtain Figure 8. It can be seen from Fig. 8 that the number of particles of the surfactant-free sample (KB group) is much higher than that of the other groups, and the number of particles of the sample added with 0.01% of the surfactant is significantly decreased when the surfactant concentration is increased. At 0.1%, the number of particles decreased to the same level as the physiological saline, and continued to increase to a concentration of 0.2%. The number of particles of the sample did not change much, and the content of the surfactant was not too large. The difference between the three surfactants is not large.
  • the content and content of the surfactant in the commercially available monoclonal antibody preparation are preferably Tween 80, and the content is made 0.1%.
  • Prescription Study-1 (40 ° C high temperature stability test): The specific composition and content are shown in Table 13 (wherein the antibody BAT1406 content is 100 mg/mL, 80 mg/mL, 50 mg/mL, respectively). After being placed at 0 °C, 1 week, 3 weeks, and 4 weeks at a temperature of 40 ° C, the main peak of SEC and the main peak of IEC and acid zone were analyzed.
  • Prescription Study-2 (4000lx Light Stability Test): The specific combination components and contents are shown in Table 13 (wherein the antibody BAT1406 content is 100 mg/mL, 80 mg/mL, 50 mg/mL, respectively).
  • the SEC main peak and the IEC main peak and acid region were analyzed on day 0, day 7, and day 14 under illumination of 4000 lx.
  • No. 1-15 in Table 13 corresponds to the serial numbers A170112-1 to A170112-15 in the figure.
  • Prescription screening study-3 for three stabilizers: arginine hydrochloride, trehalose, sorbitol, (wherein the antibody BAT1406 content is 100mg/mL, 80mg/mL, 50mg/mL, screening system
  • the remaining components are shown in Table 14).
  • pH 4.8-5.6 after the temperature of 40 ° C, after 0 days, 1 week, 3 weeks, 4 weeks, the SEC main peak, IEC main peak and acid region were analyzed; under the condition of illuminance 4000 lx, 0 was placed. After days, 1 week, and 2 weeks, the SEC main peak, IEC main peak and acid region were analyzed. The analysis results are shown in Figure 13-16.
  • the SEC data analysis showed that the purity of the SEC monomer in the three sets of stabilizer samples decreased with the decrease of the pH of the sample, and the arginine hydrochloride (Arg-HCl) sample was affected by the pH.
  • Large, trehalose (Tre) group samples are less affected by pH.
  • trehalose (Tre) is superior to sorbitan hydrochloride (Sor) in comparison to arginine hydrochloride (Arg-HCl) in terms of SEC monomer purity.
  • the purity of the SEC monomer of the arginine hydrochloride (Arg-HCl) 5.67 sample and the arginine hydrochloride (Arg-HCl) 5.44 sample was close to that of the trehalose (Tre) group and the sorbitol (Sor).
  • the SEC stability of the 4.8-group sample of the Arg-HCl group, the sorbitol (Sor) 4.77 group, and the trehalose (Tre) 4.77 group was poor, so the pH was preferably 5.0-5.6.
  • arginine hydrochloride (Arg-HCl) is superior to trehalose (Tre) and sorbitol (Sor) in terms of IEC acid content.
  • Re trehalose
  • Sor sorbitol
  • the results are shown in Figure 14d-f.
  • SEC-HPLC and IEC-HPLC are combined to control the pH, preferably between 5.0 and 5.6, and the stability of the sample is good.
  • the arginine hydrochloride (Arg-HCl) group was affected by pH, and the trehalose (Tre) group was less affected by pH.
  • trehalose (Tre) is superior to sorbitol (Sor) in comparison with arginine hydrochloride (Arg-HCl) in terms of SEC monomer purity and IEC main peak content; IEC acid region content, fine
  • the hydrochloride salt (Arg-HCl) is superior to the trehalose (Tre) and sorbitol (Sor) groups.
  • the SEC monomer purity of the arginine hydrochloride (Arg-HCl) 5.67 sample and the arginine hydrochloride (Arg-HCl) 5.44 sample was close to the trehalose (Tre) group and the sorbitol (Sor) group.
  • the IEC main peak content of the HCl hydrochloride (Arg-HCl) 5.67 and arginine hydrochloride (Arg-HCl) 5.44 samples was significantly better than the trehalose (Tre) and sorbitol (Sor) groups.
  • arginine hydrochloride Ar-HCl
  • Arg-HCl arginine hydrochloride
  • Arg-HCl arginine hydrochloride
  • the stabilizers for the monoclonal antibody preparations on the market mostly use polyols and sugars.
  • Our experimental results show that the IEC-HPLC acid of the arginine hydrochloride (Arg-HCl) group samples at high temperature (40 ° C). The content of the area is lower than that of polyols and sugars, indicating that the side is more advantageous in slowing the growth of the acid region of the sample; in addition, the purity of the SEC monomer in the arginine hydrochloride (Arg-HCl) group is under illumination (4000 lx).
  • the stability of the IEC main peak content is also superior to polyols and sugars as a whole.
  • the arginine hydrochloride group means that the stabilizer in the preparation is 3.1% arginine hydrochloride + 0.1% methionine.
  • L-929 in logarithmic growth phase was seeded in 96-well plates at 8000 cells/well/100 ul.
  • 1406 antibody and hIgG negative control group were diluted with medium containing 10% FBS and rhTNF- ⁇ (concentration 1 ng/ml) (starting concentration was 750 ng/ml, diluted at 1/3 dilution, each Two concentrations are set for each concentration, and a total of 8 gradients are established.
  • BAT1406 antibody preparation (A1, B1, C1, A2, B2, C2, A3, B3, C3 preparation, specific components are shown in Table 1) was stored at room temperature for in vitro TNF neutralization test (L- 929 bioactivity assay), stability study time is 4 weeks.
  • Freeze-Thaw Study-1 BAT1406 antibody preparations (A, B, C preparations) were formulated into three concentrations, low concentration sample LOQ (50mg/mL), medium concentration sample MOQ (80mg/mL), high concentration sample HOQ ( 100mg/mL) 3 samples (see Table 1 for specific components), taken out from the -80 °C refrigerator, placed at room temperature for more than 2h, thawed and then frozen, so cycle 5 times, the freezing time between the two melts is not Less than 12h. The concentration of the quality control after freezing and thawing was measured, and the measured value and the theoretical value were compared. The stability of the sample after 5 freeze-thaw cycles was examined. Each sample was diluted 100,000 times for Elisa detection. The recovery and precision of the duplicate well assay are listed in Table 16.
  • the recovery rate of the antibody at a high concentration of 100 mg/mL and three preparations of A, B, and C can be as high as 98% or more after repeated freeze-thaw cycles, which is not obvious because the antibody preparation is generally at a high concentration. It is more unstable, and high-concentration antibody preparations may be accompanied by negative effects such as antibody aggregation, decreased solubility, and decreased stability.
  • Freeze-thaw study-2 The pharmaceutical preparations (A1, B1, C1, A2, B2, C2, A3, B3, C3 preparations) were changed from a frozen state to a liquid state for 5 cycles, and the protein concentration was evaluated as 100 mg/ml, 80 mg/ Freeze-thaw behavior of BAT1406 antibody drug in ml, 50 mg/ml.
  • the experimental results of evaluating the effects of 5 fast and slow cold/thaw cycles from -80 ° C or -20 ° C for the preparations (A1, B1, C1, A2, B2, C2, A3, B3, C3 preparations, respectively) are shown in Table 17. .
  • the results also showed that both the fast and slow or fast freeze/thaw cycles had a smaller number of particles that could only be seen under the microscope; the in vitro neutralization TNF assay also showed that the biological activity was not affected.
  • Example 8 Microbial research
  • Microbiological studies are required on pharmaceutical formulations (A1, B1, C1, A2, B2, C2, A3, B3, C3 formulations) to determine if the formulation supports microbial growth.
  • microorganisms eg, Staphylococcus aureus, ATDD-NO.: 6538p, Candida albicans, ATDD-NO.: 10231, Aspergillus niger, ATDD-NO.: 16404, environmental isolates
  • NMT100 cfu/ml The sterile preparation is directly inoculated, and then the overall microbial growth of the inoculated preparation is examined.
  • the indicators evaluated were mainly the number of microorganisms under the microscope and the change in turbidity, wherein the lack of turbidity was an indicator of no overall growth and was detected in the inoculated container after 14 days. In addition, microorganisms cannot be re-isolated from these containers. Table 18 shows that the formulation does not support microbial growth if stored for 14 days at room temperature 20-25 °C.
  • Example 9 Subcutaneous injection metabolism in vivo
  • SD rats were injected subcutaneously with A1, B1, C1, A2, B2, C2, A3, B3, and C3 preparations to compare the drug metabolism of the three preparations in SD rats.
  • the serum of SD rats for 0 h was taken and frozen at -80 ° C for use.
  • Example 10 Recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406 protein sequence
  • a human antibody for treating TNF- ⁇ -related diseases is recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406, which is expressed by genetic engineering in CHO cells and purified by a series of standard chromatographic steps.
  • BAT1406 is an IgG antibody with a molecular weight of 148 kDa and consists of two IgG1 z, a heavy chains and two kappa light chains. Each heavy chain contains 451 amino acids, has a molecular weight of 49 kDa, and has a heavy chain amino acid sequence of SEQ ID NO. 1; each light chain contains 214 amino acids, has a molecular weight of 24 kDa, and has a light chain amino acid sequence of SEQ ID NO.
  • Example 11 Expression and purification of recombinant human anti-TNF- ⁇ monoclonal antibody BAT1406
  • Monoclonal antibody anti-TNF- ⁇ 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-k1 cell (ATCC CCL61).
  • the construction process of high-yield stable cell lines is briefly described as follows: host cells are suspended in CD-CHO medium (Gibco, CA), centrifuged in host cells in logarithmic growth phase, resuspended in fresh CD-CHO medium, and counted.
  • the 96-well plate cell culture supernatant was assayed to determine the expression level of the antibody.
  • 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. Usually 20-30 clones are transferred to shake flasks for further evaluation. Finally, 5-8 clones with the highest expression were selected for subcloning and further expression detection. The stock solution was harvested, and the cells and the medium were separated by low-speed centrifugation, and the centrifugation supernatant was centrifuged at a high speed to further clarify. Protein A affinity purification and ion exchange purification were carried out using Mab Select SuRe and Capto S manufactured by GE.
  • Example 12 Specificity study of recombinant antibody BAT1406
  • Pharmacological studies have mainly carried out in vitro pharmacodynamic tests of recombinant human anti-TNF- ⁇ monoclonal antibodies.
  • In vitro tests mainly carry out pharmacodynamic tests in the following aspects: binding ability to TNF, antibody specificity, competition for binding of TNF to receptor, inhibition of biological activity of TNF- ⁇ , and cytotoxicity experiments.
  • the specific 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: First, the rhTNF- ⁇ , rhTNF- ⁇ or rmTNF- ⁇ is diluted to 100 ng/100 ⁇ l with PBS, and the diluted rhTNF- ⁇ , rhTNF- ⁇ or rmTNF- ⁇ is added to the 96-well microtiter plate. 100 ⁇ l/well at 4 ° C overnight. The next day, it was blocked with PBS containing 3% BSA, 200 ⁇ l/well, and placed at 37 ° C for 2 hours.

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Abstract

一种靶向治疗TNF-α相关疾病的人抗体制剂，主要为液体含水药物制剂。所述液体含水药物制剂可增强含重组人抗TNF-α单克隆抗体等TNF-α拮抗剂抗体药物的稳定性，延长其在含水制剂中的保存周期。制剂在经过高温40℃保存4周及光照4000lx试验15天，至少5次循环冻融，室温放置1个月后，2～8℃保存至少36个月，仍然保持稳定。制剂可用于皮下注射使用。

Description

一种靶向治疗TNF-α相关疾病的人抗体制剂 技术领域
本发明涉及一种靶向治疗TNF-α相关疾病的抗体制剂，其主要为液体含水药物制剂。
背景技术
类风湿关节炎(RA)是一种常见的关节炎，在人群中的发病率为0.3％～1％，如不及时治疗，可导致骨破坏和关节功能障碍。在RA发病中有多种促炎性细胞因子参与，如肿瘤坏死因子-α(TNF-α)、白介素如IL-1,IL-6,IL-8等 [1]。因此，抑制促炎性细胞因子的产生或阻断其作用，有望改善关节炎症反应。近年来许多新研究开发的生物制剂可通过阻断或下调炎性细胞因子的活性而达到控制病情的进展，如TNF抑制剂、抗IL-1抗体等，这些生物制剂的主要作用机制有：(1)针对细胞因子或其受体的单克隆抗体；(2)可溶性受体拮抗剂，即不包括跨膜成分和胞内功能区的细胞因子表面受体。该受体拮抗剂可与游离的细胞因子结合，抑制后者与细胞表面受体结合。可溶性受体拮抗剂的半衰期较短，可通过加入某些整合结构如IgG的Fc受体或聚乙烷基乙二醇(polyethylene glycol,PEG)而延长；(3)受体拮抗蛋白，为无生物活性的蛋白质，可与细胞因子竞争结合细胞表面的膜受体。受体拮抗蛋白必须结合90％以上细胞表面受体方认为有效。
目前认为，在诸多RA炎症反应的细胞因子中，TNFα(Tumor Necrosis Factorα，肿瘤坏死因子-α)是最重要的促炎性细胞因子之一，TNF-α在RA病变的持续发展、局部炎症反应和组织损伤中均起着重要作用 [2]。RA中TNF-α和TNFR水平在血清、滑膜以及滑液中均显著升高，特别是病情严重和活动期患者。RA患者血清中TNF-α水平与关节损伤评分、红细胞沉降率(ESR)及贫血呈正相关。TNF-α还与RA患者体重减轻和疾病复发有关。TNF可作用于多种细胞，如促进巨噬细胞分泌炎性细胞因子和趋化因子，促进炎症反应。TNF-α的主要作用是：(1)诱导内皮细胞表达黏附分子和血管内皮生长因子(VEGF)，促进白细胞与血管内皮黏附、渗透，导致局部的炎症反应和血管VEGF生成；(2)作用于肝细胞，产生C反应蛋白(CRP)；(3)在类风湿关节炎中TNF-α可分别作用于破骨细胞、滑膜细胞和软骨细胞，导致这些细胞的活化，产生金属蛋白酶、胶原酶、基膜溶解酶(stromelysin)及前列腺素E2(PGE2)，进一步破坏软骨引起骨侵蚀、关节炎症和软骨破坏；(4)TNF-α还可促使滑膜细胞、巨噬细胞、纤维母细胞和软骨细胞产生IL-1,IL-8及TNF-α本身而加重组织损伤。因此，抑制TNF-α的作用对控制RA的病情和改善预后非常重要 [3]。
目前已被美国FDA批准的TNF-α抑制剂共有3种：可溶性受体拮抗剂-依那西普(Etanercept)，人鼠嵌合抗体-英夫利昔单抗(Infliximab)和人源化单抗。Etanercept是重组人II型肿瘤坏死因子受体-抗体融合蛋白，由II型TNF受体(p75)与IgG1的Fc部分组成二聚体；Infliximab是针对TNF的特异性IgG1单克隆抗体(由人Ig稳定区和鼠Ig可变区组成的嵌合体)；人源化单抗也是特异性针对TNF的IgG1单克隆抗体(Ig的稳定区和可变区均 为人源的)。TNF抑制剂的主要不良反应有注射部位反应、感染、肿瘤、淋巴增殖性疾病、神经脱髓鞘病变以及狼疮样综合征 [4-5]。
众所周知，药物制剂必须考虑稳定性、施用方式、浓度等因素。这些因素会影响在制造、储藏和递送过程中，抗体的物理化学降解，进而影响对施用者的风险，因此稳定的抗体制剂是必须的。此外，某些抗体制剂需要高剂量施用，需要以较高的浓度配置，而高浓度的抗体制剂可能会伴随抗体凝聚、可溶性降低、稳定性下降等负面作用。目前，制备高浓度抗体制剂仍然面临巨大的挑战。
申请人此前研发的治疗TNF-α相关疾病的人抗体BAT1406制剂，其抗体浓度为20-80mg/mL(CN103446583A)，其很难超过80mg/mL的上限。尽管存在这样的需求，然而，目前对于该抗体而言，仍没有令人满意的能支持高浓度抗体的制剂配方。
发明内容
本发明目的在于提供一种可以支持更高浓度抗体存在的制剂。
本发明的另一个目的在于提供一种浓度更高的抗体制剂。
本发明的另一个目的在于提供一种稳定性更高的治疗TNF-α相关疾病的人抗体制剂。
本发明的目的通过以下技术手段实现：
一方面，本发明提供了一种抗体制剂，其为含水药物制剂，所述的含水药物制剂包括重组人抗TNF-α单克隆抗体，缓冲液体系，稳定剂，表面活性剂。具体地，本发明的抗体制剂含有以下组分：
(1)20～120mg/ml有效量的抗TNF-a人源抗体
(2)缓冲剂
(3)0.5-10％稳定剂
(4)0.01％～0.2％表面活性剂
(5)注射用水
所述的抗体制剂的pH为4.0～7.0。
在一个实施例中，所述重组人抗TNF-α单克隆抗体是通过基因工程手段，在CHO细胞中表达的，并且通过一系列标准的层析步骤纯化获得的。在制备抗体后，再制备药物制剂。作为一种优选的实施方式，所述的重组人抗TNF-α单克隆抗体具有SEQ ID NO.1所示的重链以及SEQ ID NO.2所示的轻链(记名为BAT1406)。所述的TNF-α单克隆抗体或其抗原结合部分，能结合人TNF-α。
作为一种优选的实施方式，所述的抗体的浓度为40～120mg/ml，更为优选50～100mg/ml。
其中，所述的缓冲剂选自琥珀酸、柠檬酸、His-HCl、醋酸、磷酸盐中的一种或几种的组合；优选地，所述的缓冲剂选自His-HCl、HAc、磷酸盐和HAc的组合、His-HCl和HAc的组合；更优选His-HCl和HAc的组合。作为优选的实施方式，所述的每种缓冲剂的浓度为5～15mM；更优选为5～10mM。更优选地，当所述缓冲剂选自两种的组合时，前者 与后者在体系中的摩尔比为1:2～2:1；更优选1:1。
其中，所述的稳定剂为0.5％～5.0％Arg-HCl和0.01％～2％Met；或者选自3.0％～6.0％山梨醇；又或者选自7％～10％海藻糖。
其中，所述的表面活性剂选自吐温80、吐温20或泊洛沙姆188。
本制剂所选的表面活性剂均属于非离子型表面活性剂，它们对制剂的稳定性有提升作用，特别是在振荡和反复冻融的条件下。发明人发现，在本发明制剂中，不同种类的非离子表面活性剂对于稳定性的提升作用相差不大。如吐温80、吐温20、泊洛沙姆188均属于非离子型表面活性剂，它们在生物单抗制剂中的用量为0.001％--0.1％，使用量很低，极少量的表面活性剂即可满足样品稳定性需求。这可能是因为：表面活性剂是具有亲水基团和亲油基团的两亲分子，通过分子中不同部分分别对于两相的亲和，使两相均将其看作本相的成分，分子排列在两相之间，使两相的表面相当于转入分子内部，从而降低表面张力。由于两相都将其看作本相的一个组分，就相当于两个相与表面活性剂分子都没有形成界面，通过这种方式部分地消灭了两个相的界面，因此降低了表面张力和表面自由能，从而达到稳定单抗蛋白的目的。
作为优选的实施方式，本发明的抗体制剂含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)2％～3.5％Arg-HCl
(5)0.01％～0.5％Met
(6)0.01％～0.1％Tween 80
(7)注射用水
其中，所述制剂的pH为5.0～5.6，优选地，pH为5.2～5.6；
或者优选地，含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)4.0％～5％山梨醇
(5)0.01％～0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.0～5.6，优选地，pH为5.0～5.4；
又或者优选地，含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)7％～9％海藻糖
(5)0.01％～0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.0～5.6，优选地，pH为5.0～5.4。
作为一种优选的实施方案，本发明的抗体制剂含有以下组分：
(1)80～120mg/ml的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)2％～3.5％Arg-HCl
(5)0.01％～0.5％Met
(6)0.01％～0.1％Tween 80
(7)注射用水
其中，所述制剂的pH为5.0～5.6，优选地，pH为5.2～5.6；
或者优选地，含有以下组分：
(1)80～120mg/ml的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)4.0％～5.0％山梨醇
(5)0.01％～0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.0～5.6，优选地，pH为5.0～5.4；
又或者优选地，含有以下组分：
(1)80～120mg/ml有效量的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
(2)5～10mM HAc
(3)5～10mM His-HCl
(4)7％～9％海藻糖
(5)0.01％～0.1％Tween 80
(6)注射用水
所述的制剂的pH为5.0～5.6，优选地，pH为5.0～5.4。
或者，作为更优选的实施方式，所述的制剂含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)3.1％Arg-HCl
(5)0.1％Met
(6)0.1％Tween 80
(7)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.4±0.2；
或者更优选地，含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)4.2％山梨醇
(5)0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.2±0.2；
又或者更优选地，含有以下组分：
(1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)8.4％海藻糖
(5)0.1％Tween 80
(6)注射用水
所述的制剂的pH为5.3±0.3，优选地，pH为5.2±0.2。
在本发明优选的一个实施例中，所述的制剂含有以下组分：
(1)100mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)3.1％Arg-HCl
(5)0.1％Met
(6)0.1％Tween 80
(7)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.4±0.2；
在本发明优选的另一个实施例中，所述的制剂含有以下组分：
(1)50mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)3.1％Arg-HCl
(5)0.1％Met
(6)0.1％Tween 80
(7)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.4±0.2；
在本发明优选的另一个实施例中，所述的制剂含有以下组分：
(1)100mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)4.2％山梨醇
(5)0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.2±0.2；
在本发明优选的另一个实施例中，所述的制剂含有以下组分：
(1)50mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)4.2％山梨醇
(5)0.1％Tween 80
(6)注射用水
其中，所述制剂的pH为5.3±0.3，优选地，pH为5.2±0.2；
在本发明优选的另一个实施例中，所述的制剂含有以下组分：
(1)100mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)8.4％海藻糖
(5)0.1％Tween 80
(6)注射用水
所述的制剂的pH为5.3±0.3，优选地，pH为5.2±0.2。
在本发明优选的另一个实施例中，所述的制剂含有以下组分：
(1)50mg/ml的抗TNF-ɑ人源抗体
(2)5mM HAc
(3)5mM His-HCl
(4)8.4％海藻糖
(5)0.1％Tween 80
(6)注射用水
所述的制剂的pH为5.3±0.3，优选地，pH为5.2±0.2。
本发明的制剂还含有NaOH，用于调节pH。
本发明的制剂的渗透压为150～400mOsm/Kg；作为优选的实施方式，渗透压为260～340mOsm/Kg。
另一方面，本发明还提供了上述抗体制剂的制备方法，其包含以下步骤：
⑴配制包含稳定剂的制剂缓冲液；
⑵采用UF/DF超滤方法，使用Millipore超滤膜包(再生纤维素，30kDa分子量截留)，对原料药(如抗体BAT1406)进行超滤换液，换液至少8个体积(即被换液的样品的即时体积)的制剂缓冲液，检测超滤系统透过端pH和电导，当透过端的pH、电导与刚配制的制剂缓冲液的pH、电导一致时，说明换液充分，可进入下一步，浓缩。UF/DF系统即切换至超滤浓缩模式，且制剂被浓缩至目标浓度水平。
⑶在完成UF/DF步骤之后，添加特定量的表面活性剂以达到所要求表面活性剂的浓度。
⑷在填充主要容器之前，制剂在超净工作台中使用0.2微米过滤器进行无菌过滤。填充也是无菌进行。
本发明的含水液体抗体制剂适用于进行皮下注射。
本发明的抗体制剂中，重组人抗TNF-α单克隆抗体BAT1406的浓度可高达120mg/ml，例如，抗体可在20～120mg/ml的浓度范围下均稳定存在，而CN103446583A中BAT1406抗体制剂中抗体浓度最高仅能达到80mg/ml(CN103446583A)，本发明将其上限提高了50％。
难得的是，在抗体高浓度的情况下，本发明的抗体制剂还具有很高的稳定性，能够延长BAT1406在含水制剂中的保存周期；具体表现在，该制剂在室温下保持稳定至少1个月；在2-8℃下至少能稳定36个月；至少在5次循环冻融后保持稳定性。更具体地，BAT1406在含水制剂中在室温下保持稳定1个月；在2-8℃下36个月；在5次循环冻融后，EC50在22ng/ml以下，更优选21ng/ml以下。
本发明所开发的重组人抗TNF-α单克隆抗体制剂，其中所含的高浓度抗体蛋白更加稳定，更加耐受高温变化引起的聚集反应，因此本发明的抗体制剂可用于保存靶向治疗TNF-α相关的人抗体，增强含重组人抗TNF-α单克隆抗体等TNF-α拮抗剂抗体药物的稳定性，延长其在含水制剂中的保存周期，对于靶向治疗TNF-α相关疾病有重要意义。
附图说明
图1a缓冲体系筛选SEC主峰分析
图1b缓冲体系筛选SEC片段(聚体)分析
图2a缓冲体系筛选IEC主峰分析
图2b缓冲体系筛选IEC酸峰分析
图3a缓冲体系筛选CE(NR)主峰分析
图3b缓冲体系筛选CE(NR)碎片(片段)分析
图4a 100mg/ml制剂组高温(40℃)SEC主峰变化
图4b 80mg/ml制剂组高温(40℃)SEC主峰变化
图4c 50mg/ml制剂组高温(40℃)SEC主峰变化
图5a 100mg/ml制剂组高温(40℃)IEC主峰变化
图5b 80mg/ml制剂组高温(40℃)IEC主峰变化
图5c 50mg/ml制剂组高温(40℃)IEC主峰变化
图5d 100mg/ml制剂组高温(40℃)IEC酸峰变化
图5e 80mg/ml制剂组高温(40℃)IEC酸峰变化
图5f 50mg/ml制剂组高温(40℃)IEC酸峰变化
图6a 100mg/ml制剂组光照SEC主峰变化
图6b 80mg/ml制剂组光照SEC主峰变化
图6c 50mg/ml制剂组光照SEC主峰变化
图7a 100mg/ml制剂组光照IEC主峰变化
图7b 80mg/ml制剂组光照IEC主峰变化
图7c 50mg/ml制剂组光照IEC主峰变化
图7d 100mg/ml制剂组光照IEC酸峰变化
图7e 80mg/ml制剂组光照IEC酸峰变化
图7f 50mg/ml制剂组光照IEC酸峰变化
图8不同浓度表面活性剂样品稀释后的不溶性微粒数(≥10μm)(个/mL)
图9a不同辅料处方40℃高温对抗体(100mg/ml)稳定性的影响(SEC主峰)
图9b不同辅料处方40℃高温对抗体(80mg/ml)稳定性的影响(SEC主峰)
图9c不同辅料处方40℃高温对抗体(50mg/ml)稳定性的影响(SEC主峰)
图10a不同辅料处方40℃高温对抗体(100mg/ml)稳定性的影响(IEC主峰)
图10b不同辅料处方40℃高温对抗体(80mg/ml)稳定性的影响(IEC主峰)
图10c不同辅料处方40℃高温对抗体(50mg/ml)稳定性的影响(IEC主峰)
图10d不同辅料处方40℃高温对抗体(100mg/ml)稳定性的影响(IEC酸峰
图10e不同辅料处方40℃高温对抗体(80mg/ml)稳定性的影响(IEC酸峰)
图10f不同辅料处方40℃高温对抗体(50mg/ml)稳定性的影响(IEC酸)
图11a不同辅料处方4000lx光照对抗体(100mg/ml)稳定性的影响(SEC主峰)
图11b不同辅料处方4000lx光照对抗体(80mg/ml)稳定性的影响(SEC主峰)
图11c不同辅料处方4000lx光照对抗体(50mg/ml)稳定性的影响(SEC主峰)
图12a不同辅料处方4000lx光照对抗体(100mg/ml)稳定性的影响(IEC主峰)
图12b不同辅料处方4000lx光照对抗体(80mg/ml)稳定性的影响(IEC主峰)
图12c不同辅料处方4000lx光照对抗体(50mg/ml)稳定性的影响(IEC主峰)
图12d不同辅料处方4000lx光照对抗体(100mg/ml)稳定性的影响(IEC酸峰)
图12e不同辅料处方4000lx光照对抗体(80mg/ml)稳定性的影响(IEC酸峰)
图12f不同辅料处方4000lx光照对抗体(50mg/ml)稳定性的影响(IEC酸峰)
图13a抗体(100mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(高温40℃)
图13b抗体(80mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(高温40℃)
图13c抗体(50mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(高温40℃)
图14a抗体(100mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(高温40℃)
图14b抗体(80mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(高温40℃)
图14c抗体(50mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(高温40℃)
图14d抗体(100mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(高温40℃)
图14e抗体(80mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(高温40℃)
图14f抗体(50mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(高温40℃)
图15a抗体(100mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(光照4000lx)
图15b抗体(80mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(光照4000lx)
图15c抗体(50mg/ml)分别在三种稳定剂的不同PH值下SEC主峰变化情况(光照4000lx)
图16a抗体(100mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(光照4000lx)
图16b抗体(80mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(光照4000lx)
图16c抗体(50mg/ml)分别在三种稳定剂的不同PH值下IEC主峰变化情况(光照4000lx)
图16d抗体(100mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(光照4000lx)
图16e抗体(80mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(光照4000lx)
图16f抗体(50mg/ml)分别在三种稳定剂的不同PH值下IEC酸峰变化情况(光照4000lx)
图17a制剂A1室温放置第0天与第4周EC50对比
图17b制剂B1室温放置第0天与第4周EC50对比
图17c制剂C1室温放置第0天与第4周EC50对比
图17d制剂A2室温放置第0天与第4周EC50对比
图17e制剂B2室温放置第0天与第4周EC50对比
图17f制剂C2室温放置第0天与第4周EC50对比
图17g制剂A3室温放置第0天与第4周EC50对比
图17h制剂B3室温放置第0天与第4周EC50对比
图17i制剂C3室温放置第0天与第4周EC50对比
图18a重组人抗TNF-α单克隆抗体BAT1406的A1、B1、C1三种制剂的皮下注射体内代谢过程
图18b重组人抗TNF-α单克隆抗体BAT1406的A2、B2、C2三种制剂的皮下注射体内代谢过程
图18c重组人抗TNF-α单克隆抗体BAT1406的A3、B3、C3三种制剂的皮下注射体内代谢过程
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
需要说明的是，本发明中，除非特别说明的之外，所涉及到组分的“％”是指质量体积(w/v)百分比，例如，某组分含量6％，即指100ml制剂体系中含6g该组分。
本文的“稳定性”、“稳定”，是指包含抗体(包括其抗体片段)的液体制剂中，抗体(包括其抗体片段)在给定的生产、制备、运输和/或贮存条件下不发生、或仅极少地发生聚集、降解或片段化。“稳定”制剂在给定的生产、制备、运输和/或贮存条件下保持生物学活性。可通过例如SEC-HPLC、IEC-HPLC、CE-SDS等技术测量的所述制剂的聚集、降解或片段化程度等，从而评估所述抗体(包括其抗体片段)的稳定性。
缓冲液是由缓冲剂配置而成，能起到在一定程度上保持溶液的pH值相对稳定的作用。常见的缓冲剂例如，有机酸盐诸如柠檬酸、抗坏血酸、葡萄酸、碳酸、酒石酸、琥珀酸、乙酸或邻苯二甲酸的盐；Tris，氨丁三醇盐酸盐、或磷酸盐；氨基酸组分缓冲剂如甘氨酸、组氨酸等。常见的缓冲液体系例如：邻苯二甲酸盐-盐酸盐缓冲液、磷酸氢二钠-柠檬酸缓冲液、柠檬酸-氢氧化钠-盐酸缓冲液、柠檬酸-柠檬酸钠缓冲液、醋酸-醋酸钠缓冲液、磷酸二氢钾-氢氧化钠缓冲液、磷酸盐缓冲液(磷酸氢二钠-磷酸二氢钠缓冲液)、巴比妥钠-盐酸缓冲液、Tris-HCl缓冲液、硼酸-硼砂缓冲液、甘氨酸-氢氧化钠缓冲液、甘氨酸-盐酸缓 冲液、硼砂-氢氧化钠缓冲液、碳酸钠-碳酸氢钠缓冲液，等等。
从理论上讲上述的缓冲液体系均可适用于本发明的抗体制剂，用以维持体系pH值的稳定。不过，从实践上看，缓冲液的选择仍然会较为关键地影响着本发明抗体制剂的稳定性。尤其是在高浓度抗体存在的情况下，缓冲体系的选择显得更为重要。
本发明经过实验筛选出一系列较为适合的缓冲液体系，为：琥珀酸缓冲液、柠檬酸缓冲液、组氨酸缓冲液、醋酸缓冲液、磷酸盐缓冲液+柠檬酸缓冲液、磷酸盐缓冲液+醋酸缓冲液、组氨酸缓冲液+琥珀酸缓冲液、组氨酸缓冲液+柠檬酸缓冲液、组氨酸缓冲液+醋酸缓冲液、琥珀酸缓冲液+柠檬酸缓冲液、琥珀酸缓冲液+醋酸缓冲液。而其中，最为优选的缓冲液体系为组氨酸缓冲液+醋酸缓冲液，实验发现，缓冲液中组氨酸组分能提高样品的SEC和IEC稳定性，醋酸组分在不影响样品稳定性的基础上，能够为缓冲体系提供一个稳定的pH缓冲范围，因此优选组氨酸缓冲液+醋酸缓冲液。
组氨酸是一种碱性氨基酸，制备组氨酸缓冲液时，作为非限制性的例子，可以将组氨酸溶入水后，用盐酸调pH，即形成组氨酸缓冲液；其中，加入的盐酸与一部分组氨酸反应形成组氨酸盐酸盐，因此，在该缓冲液中，存在着组氨酸和组氨酸盐酸盐。不同pH的组氨酸缓冲液则影响了其中组氨酸和组氨酸盐酸盐的比例不同，所述的组氨酸的摩尔浓度是组氨酸和组氨酸盐酸盐两者之和。因此，在本发明中，组氨酸缓冲液也表示为缩写“His-HCl”。
本制剂中稳定剂有调节渗透压的作用，故该稳定剂也可称作渗透压调节剂。
具体而言，本发明所提供的制剂为pH4.0～7.0的液体含水药物，该制剂含有20～120mg/ml浓度的抗体，并且具有增强含重组人抗TNF-α单克隆抗体等TNF-α拮抗剂抗体药物的稳定性作用。所述制剂的抗体浓度低于20mg/ml也可以，不过根据给药量，需要增加体积，为了减少给药体积和给药次数，以减少病人疼痛感，控制给药次数多带来的感染风险，故提高浓度是优选。本发明的制剂包括以下成分：能以高亲和力、低解离速度、高中和能力结合人hTNF-α的抗体；缓冲液，包括His-HCl，或者HAc、或者磷酸盐和HAc的组合、或者His-HCl和HAc的组合；渗透压调节剂，包括山梨醇、海藻糖、精氨酸和甲硫氨酸中的一种或两种；表面活性剂，包括Tween 80；调节pH的氢氧化钠和注射用水。
在一些实施例中，所述抗体在所述液体含水药物制剂中的浓度为大约40～120mg/ml，优选为45～110mg/ml。所述抗体是重组人抗TNF-α单克隆抗体BAT1406，BAT1406为阿达木单抗生物类似药。
本发明提供了不同缓冲体系的含水抗体制剂，包括琥珀酸缓冲液、柠檬酸缓冲液、组氨酸缓冲液、醋酸缓冲液、磷酸盐缓冲液+柠檬酸缓冲液、磷酸盐缓冲液+醋酸缓冲液、组氨酸缓冲液+琥珀酸缓冲液、组氨酸缓冲液+柠檬酸缓冲液、组氨酸缓冲液+醋酸缓冲液、琥珀酸缓冲液+柠檬酸缓冲液、琥珀酸缓冲液+醋酸缓冲液，pH为大约3.0～6.0。
本发明提供液体含水抗体制剂，所述制剂包括稳定剂(多元醇)，以便调节液体体系的渗透压，稳定所述抗体。将所述稳定剂添加到所述抗体中，其用量可以根据需要的所述制剂的等渗而改变。在本发明制剂的优选方案中，被作为渗透压调节剂用在所述制剂中的是精氨酸盐酸盐和甲硫氨酸，在本发明的优选方案中，所述稳定剂选自3.0％～6.0％山梨醇， 优选山梨醇的浓度为4.2％；或选自3.5％～5.5％甘露醇，优选甘露醇的浓度为4.5％；或0.9％氯化钠；或1.0％～5.0％脯氨酸，优选脯氨酸的浓度为2.5％；或3.5％～9％蔗糖，优选蔗糖的浓度为6％；或7.0％～10％海藻糖，优选海藻糖的浓度为8.4％；或0.5％～5.0％精氨酸盐酸盐，优选精氨酸盐酸盐的浓度为3.1％；或0.1％～2％甲硫氨酸。所述制剂的渗透压范围为150～400mOsm/Kg。
本发明提供液体含水抗体制剂，所述制剂包括缓冲体系。所述缓冲体系包括组氨酸缓冲液、醋酸缓冲液或磷酸盐缓冲液。在本发明的制剂的一种优选方案中，作为缓冲体系的是组氨酸缓冲液和醋酸缓冲液，组氨酸缓冲液和醋酸缓冲液的浓度范围均为5～15mM，优选5～10mM，更优选浓度均为5mM。在本发明制剂的另一种优选方案中，作为缓冲体系的是组氨酸缓冲液、醋酸缓冲液及磷酸缓冲液。本方案中的一种缓冲液是组氨酸缓冲液，浓度范围是5～15mM，优选浓度为5mM。本方案中的另一种缓冲液是醋酸缓冲液，浓度范围是10～25mM，优选浓度为17mM。本方案中的另一种缓冲液是磷酸缓冲液，浓度范围是5～15mM，优选浓度为10mM。在本发明制剂的另一种优选方案中，作为缓冲体系的是组氨酸缓冲液及醋酸缓冲液。本方案中的一种缓冲液是组氨酸缓冲液，浓度范围是15～25mM，优选浓度为20mM。本方案中的另一种缓冲液是醋酸缓冲液，浓度范围是10～25mM，优选浓度为17mM。在本发明制剂的另一种优选方案中，作为缓冲体系的是醋酸缓冲液及磷酸缓冲液。本方案中的一种缓冲液是醋酸缓冲液，浓度范围是10～25mM，优选浓度为17mM。本方案中的另一种缓冲液是磷酸缓冲液，浓度范围是5～15mM，优选浓度为10mM。
本发明提供液体含水抗体制剂，所述制剂包括表面活性剂。典型的表面活性剂包括非离子表面活性剂，如Tween 80。表面活性剂能减少所述制备的抗体的聚集和/或减少颗粒在所述制剂中的形成和/或减少吸附。在本发明制剂的一种可选的实施方案中，所述制剂以Tween 80为表面活性剂。在优选方案中，所述制剂包括大约含0.01％～0.2％的Tween80，更优选0.01％～0.1％，更优选地，在本发明的制剂中均存在大约0.1％的Tween 80。
在本发明的一个优选的实施方案中，本发明提供的液体含水抗体制剂组合方案中，所述制剂缓冲体系包括组氨酸缓冲液和醋酸缓冲液。所述制剂的稳定剂包括山梨醇、或者海藻糖、或者精氨酸盐酸盐和甲硫氨酸的组合。所述制剂的表面活性剂包括Tween 80。所述制剂的pH值范围在4.0～7.0之间。所述制剂的渗透压范围在150～400mOsm/Kg之间。在本发明的一种方案中，被作为缓冲体系的是组氨酸缓冲液及醋酸，浓度范围均为5～15mM，优选5～10mM，更优选浓度均为5mM，pH值范围在4.0～7.0，优选pH值为5.0-5.6，更优选pH值为5.2～5.6。被作为稳定剂的精氨酸盐酸盐和甲硫氨酸，浓度范围分别为0.5％～5.0％、0.01％～2％，优选浓度分别为3.1％和0.1％。被作为表面活性剂的是Tween 80，其浓度范围是0.01％～0.2％，优选浓度是0.1％。所述制剂的渗透压范围是150～400mOsm/Kg，优化渗透压为260～340mOsm/Kg。在本发明制剂的另一种优选方案中，被作为缓冲体系的是组氨酸缓冲液及醋酸，浓度范围均为5～15mM，优选5～10mM，更优选浓度均为5mM，pH值范围在4.0～7.0，优选pH值范围为5.0～5.6，更优选pH值范围为5.0～5.4。被作为稳定剂的山梨醇，浓度范围分别为3％～6％，优选浓度为4.2％。被作为表面活性剂的是Tween 80，其浓度范围是0.01％～0.5％，优选浓度是0.1％。所述制剂的渗透压范围为 150～400mOsm/Kg，优选渗透压为260～340mOsm/Kg。在本发明制剂的另一种优选方案中，被作为缓冲体系的是组氨酸缓冲液及醋酸，浓度范围均为5～15mM，优选5～10mM，更优选浓度均为5mM，pH值范围在4.0～7.0，优选pH值为5.0～5.6，更优选pH值为5.0～5.4。被作为稳定剂的是海藻糖，浓度范围分别为7％～10％，优选浓度为8.4％。被作为表面活性剂的是Tween 80，其浓度范围是0.01％～0.2％，优选浓度是0.1％。所述制剂的渗透压为150～400mOsm/Kg，优选渗透压为260～340mOsm/Kg。
本发明提供液体含水药物制剂，包括适合治疗用途的抗体，这种药物制剂便于施用，并且含有高蛋白浓度，主要用于治疗由TNF-α引起的病症。在一种实施方案中，所述药物制剂具有增强稳定性的作用。在另一种实施方案中，本发明的制剂在经过至少5次冻融循环后是稳定的。在另一种实施方案中，本发明的制剂，经过室温中放置1个月后保持稳定。在另一种实施方案中，本发明的制剂，在2～8℃中至少能保持36个月的稳定。在另一种实施方案中，所述抗体针对人TNF-α。在另一种实施方案中，所述抗体是重组人抗TNF-α单克隆抗体BAT1406。
在本发明的另一种实施方案中，所述液体含水药物制剂包括抗体或其抗原结合部分，它是重组抗体或其抗原结合部分，它能抑制人TNF-α诱导的ELAM-1在人静脉内皮细胞上的表达。在另一种实施方案中，所述制剂包括重组人抗TNF-α单克隆抗体BAT1406，其具有2条SEQ ID NO.1所示的重链以及2条SEQ ID NO.2所示的轻链。
本发明提供含水的药用制剂，包括有效量的抗体成分，缓冲体系是组氨酸缓冲液与醋酸缓冲液，渗透压稳定剂为精氨酸盐酸盐与甲硫氨酸、或山梨醇、或海藻糖，表面活性剂Tween 80和注射用水，pH为大约4.0～7.0。在本发明的一种实施方案中，所述制剂适合于进行皮下注射。在本发明的一种实施方案中，所述抗体在所述液体含水药物制剂中的浓度为大约100mg/ml；在本发明的一种实施方案中，所述抗体在所述液体含水药物制剂中的浓度为大约80mg/ml；在本发明的另一种实施方案中，所述抗体在所述液体含水药物制剂中的浓度为大约50mg/ml。
在本发明的优选实施方案中，所述制剂是含有表1a-1c所示成分的溶液；优选地，所述制剂是装在预灌封注射器中。其中制剂包含的成份可以为40mg有效量的抗hTNF-α抗体；3.1％精氨酸盐酸盐与0.1％甲硫氨酸，0.1％Tween 80，5mM组氨酸盐酸盐及5mM醋酸，用于调节pH的氢氧化钠和注射用水(见表1a)。其中制剂包含的成份还可以为40mg有效量的抗hTNF-α抗体；4.2％山梨醇，0.1％Tween 80，5mM组氨酸盐酸盐及5mM醋酸，用于调节pH的氢氧化钠和注射用水(见表1b)。其中制剂包含的成份还可以为40mg有效量的抗hTNF-α抗体；8.4％海藻糖，0.1％Tween 80，5mM组氨酸盐酸盐及5mM醋酸，用于调节pH的氢氧化钠和注射用水(见表1c)。
表1a.重组人抗TNF-α单克隆抗体BAT1406的A1制剂成份列表(抗体浓度100mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
精氨酸盐酸盐 3.1％ 渗透压调节剂
甲硫氨酸 0.1％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至400ul 溶剂
表1b.重组人抗TNF-α单克隆抗体BAT1406的B1制剂成份列表(抗体浓度100mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
山梨醇 4.2％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至400ul 溶剂
表1c.重组人抗TNF-α单克隆抗体BAT1406的C1制剂成份列表(抗体浓度100mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
海藻糖 8.4％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至400ul 溶剂
在本发明的另一优选实施方案中，所述制剂含有表1d-1f所示成分的溶液；优选地，所述制剂是装在预灌封注射器中。
表1d.重组人抗TNF-α单克隆抗体BAT1406的A2制剂成份列表(抗体浓度80mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
精氨酸盐酸盐 3.1％ 渗透压调节剂
甲硫氨酸 0.1％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至500ul 溶剂
表1e.重组人抗TNF-α单克隆抗体BAT1406的B2制剂成份列表(抗体浓度80mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
山梨醇 4.2％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至500ul 溶剂
表1f重组人抗TNF-α单克隆抗体BAT1406的C2制剂成份列表(抗体浓度80mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
海藻糖 8.4％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至500ul 溶剂
在本发明的另一优选实施方案中，所述制剂含有表1g-1i所示成分的溶液；优选地，所述制剂是装在预灌封注射器中。
表1g.重组人抗TNF-α单克隆抗体BAT1406的A3制剂成份列表(抗体浓度50mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
精氨酸盐酸盐 3.1％ 渗透压调节剂
甲硫氨酸 0.1％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至800ul 溶剂
表1h.重组人抗TNF-α单克隆抗体BAT1406的B3制剂成份列表(抗体浓度50mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
山梨醇 4.2％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至800ul 溶剂
表1i重组人抗TNF-α单克隆抗体BAT1406的C3制剂成份列表(抗体浓度50mg/mL)
成分名称 用量 功能
活性物质：抗体BAT1406 40mg 活性物质
海藻糖 8.4％ 渗透压调节剂
组氨酸盐酸盐 5mM 缓冲剂
醋酸 5mM 缓冲剂
Tween80 0.1％ 表面活性剂
氢氧化钠 调pH至5.3±0.3 pH调节剂
注射用水 补充至800ul 溶剂
综上而言，本发明提供液体含水药物，作为一种实施方式，该制剂包含的成份可以为40～120mg/ml的抗体浓度，5～15mM组氨酸缓冲液，5～15mM醋酸缓冲液，0.5％～5.0％精氨酸盐酸盐，0.01％～2％甲硫氨酸，0.01％～0.2％Tween 80和注射用水，pH为4.0～7.0。优选方案包括45～110mg/ml的抗体浓度，5mM组氨酸缓冲液，5mM醋酸缓冲液，3.1％精氨酸盐酸盐，0.1％甲硫氨酸，0.1％Tween80和注射用水，pH为5.3±0.3，更优选pH为5.4±0.2，调节pH的氢氧化钠和注射用水(以下简称为A制剂)。
作为另外一种实施方式，该制剂包含的成份可以为40～120mg/ml的抗体浓度，5～15mM组氨酸缓冲液，5～15mM醋酸缓冲液，3.0％～6.0％山梨醇，0.01％～0.2％Tween 80和注射用水，pH为4.0～7.0。优选方案包括45～110mg/ml的抗体浓度，5mM组氨酸缓冲液，5mM醋酸缓冲液，4.2％山梨醇，0.1％Tween 80和注射用水，pH为5.3±0.3，更优选pH为5.2±0.2，调节pH的氢氧化钠和注射用水(以下简称为B制剂)。
作为另外一种实施方式，该制剂包含的成份可以为40～120mg/ml的抗体浓度，5～15mM组氨酸缓冲液，5～15mM醋酸缓冲液，7％～10％海藻糖，0.01％～0.2％Tween 80和注射用水，pH为4.0～7.0。优选方案包括45～110mg/ml的抗体浓度，5mM组氨酸缓冲液，5mM醋酸缓冲液，8.4％海藻糖，0.1％Tween 80和注射用水，pH为5.3±0.3，更优选pH为5.2±0.2，调节pH的氢氧化钠和注射用水(以下简称为C制剂)。
本发明的制剂在经过至少5次冻融循环后是稳定的。该含水药物制剂能够在2～8℃下放置36个月而保持稳定。所述液体含水药物制剂主要用于治疗由TNF-α引起的病症。所述制剂适合皮下注射。所述液体含水药物制剂可增强含重组人抗TNF-α单克隆抗体等TNF-α拮抗剂抗体药物的稳定性，延长其在含水制剂中的保存周期。
注：组氨酸盐酸盐、组氨酸缓冲液、组氨酸、His-HCl四者在本文中可理解为同一含义，即采用组氨酸和盐酸配置成的缓冲液。
实施例
实施例1：缓冲体系对制剂稳定性的影响
11组含有抗体的缓冲体系组合如表2。
11组含有抗体的缓冲体系的制备方法为：
样品组分为：10mM缓冲液加上BAT1406抗体，抗体浓度为50mg/mL。因本实验是筛选缓冲液，故暂未加入其它成分。
制备方法：称取10mM的缓冲剂，溶于水中后，用氢氧化钠调节pH至5.2，定容。最后采用超滤的方法，制备出11组样品。超滤方法，前文有叙述。
在一定的pH范围内，分别在第0、5、10、15天时，进行重组人抗TNF-α单克隆抗体BAT1406稳定性的研究，分别进行了SEC主峰、聚体和片段的分析；IEC主峰及酸区的分析；CE(NR)主峰及碎片的分析；DSC检测。
SEC-HPLC的分析方法：
1，准备液相色谱系统和TOSOH生物科技TSK-GEL G3000SWXL色谱柱(柱规格为7.8*300mm，5μm)。开启液相色谱仪并打开仪器操作软件，调用方法，打开泵进流动相，设置紫外检测器波长，柱温设定为室温，调流速0.5ml/min，平衡30-60min至基线平稳。
2，进样，记录色谱图，积分后按照峰面积归一法计算供试液的单体或多聚体百分含量。
IEC-HPLC的分析方法：
1，准备液相色谱系统和ProPac WCX-10弱酸阳离子交换分离柱。开启电源，打开液相数据分析系统，调用方法，打开泵，以94％流动相A，0.2ml/min初始条件平衡色谱柱，待压力稳定后调流速至0.8ml/min平衡30-60min至基线平稳。
2，进样，对检测的色谱图进行手动积分，按峰面积归一法计算三个赖氨酸变体(lys0、lys1和lys2)总的百分含量。
CE-SDS(NR)的分析方法：
毛细管电泳仪采用Beckman的PA800plus，样品进样后，记录色谱图，积分处理数据，采用面积归一化方法计算单体峰的百分含量。
DSC的分析方法：
1，开机预热，清洗管路，样品稀释，脱气，进样。
2，打开电脑桌面上的操作程序，设定好参数，进行检测。
3，对检测的峰图进行拟合分析。
表2各组缓冲体系组分
Figure PCTCN2019074715-appb-000001
注：表1序号1～11分别对应图中序号A160923-1～A160923-11
结果显示，从SEC主峰数据作图可以看出，3、4、6、7、9号处方的主峰下降幅度 较少，稳定性较好；2、5、8、10号处方的主峰下降较大，稳定性差，结果见图1a；从SEC片段数据作图可以看出，3、4、6、9、11号处方的SEC片段增加较少，2、5、8、10号处方的SEC片段增加较多，结果见图1b。
从IEC主峰数据作图可以看出，3、4号处方的IEC主峰下降幅度较少，稳定性较好；1、2、10、11号处方的主峰下降较多，稳定性较差，结果见图2a；从IEC酸区数据作图可以看出，3、5号处方的酸区增加较少，1、10、11号处方的酸区增加较多，结果见图2b。
从CE(NR)的主峰数据看，3、4、6、9号样品的主峰下降较小，1、2、5、8、10号样品的主峰下降较多，结果见图3a；从CE(NR)的Fraction数据看，3、4、6、9号样品的Fraction增长较小，2、5、8、10号样品的Fraction增加较多，结果见图3b。
从DSC的Tm值数据看，4、6、9、11号样品的Tm值较高，1、2、3、8、10号样品的Tm值较低，具体结果见表3。
表3样品DSC*的Tm值
样品名称 缓冲体系 Tm(℃)
1 琥珀酸缓冲液 73.59
2 柠檬酸缓冲液 72.68
3 组氨酸缓冲液 73
4 醋酸缓冲液 74.51
5 磷酸缓冲液+柠檬酸缓冲液 73.63
6 磷酸缓冲液+醋酸缓冲液 74.42
7 组氨酸缓冲液+琥珀酸缓冲液 73.44
8 组氨酸缓冲液+柠檬酸缓冲液 72.71
9 组氨酸缓冲液+醋酸缓冲液 73.84
10 琥珀酸缓冲液+柠檬酸缓冲液 72.99
11 琥珀酸缓冲液+醋酸缓冲液 73.94
*＝差示扫描量热法
综上所述，从SEC主峰、碎片，IEC主峰、酸区，CE(NR)主峰、碎片和DSC的Tm值对比结果分析，3、4、6、9号缓冲体系可选，具体情况见表4。
表4可选缓冲体系成分及pH范围
序号 缓冲体系 pH范围
3 组氨酸缓冲液 5.5～6.5
4 醋酸缓冲液 3.6～5.8
6 磷酸缓冲液+醋酸缓冲液 4.0～6.0
9 组氨酸缓冲液+醋酸缓冲液 4.0～6.0
实施例2：不同缓冲剂组合对抗体稳定性的影响
按表5的成分，制备BAT1406抗体制剂，置于40℃条件下，放置4周后，对样品分别进行SEC-HPLC主峰、多聚体、片段及IEC-HPLC主峰、酸区、碱峰、碱峰2、碱峰3分析。
表5各处方组成成分表
Figure PCTCN2019074715-appb-000002
上述制剂样品的制备方法为：
制备方法，其包含以下步骤：
⑴配制缓冲液。以分别配制1L为例，1至4号缓冲剂组合配制方法依次分别为：称取1.05g组氨酸盐酸盐、0.834g醋酸，溶于水，用氢氧化钠调pH至5.2，定容至1L；称取2.10g组氨酸盐酸盐、2.836g醋酸、1.38g磷酸氢二钠，溶于水，用氢氧化钠调pH至5.2，定容至1L；称取4.19g组氨酸盐酸盐、2.836g醋酸，溶于水，用氢氧化钠调pH至5.2，定容至1L；称取2.836g醋酸、1.38g磷酸氢二钠，溶于水，用氢氧化钠调pH至5.2，定容至1L。
⑵采用UF/DF超滤方法，使用超滤膜包(再生纤维素，30kDa分子量截留)，对原料药(如抗体BAT1406)进行超滤换液，换液至少8个体积(即被换液的样品的即时体积)的制剂缓冲液，检测超滤系统透过端pH和电导，当透过端的pH、电导与刚配制的制剂缓冲液的pH、电导一致时，说明换液充分，可进入下一步，浓缩。UF/DF系统即切换至超滤浓缩模式，且制剂被浓缩至目标浓度水平。
(3)在填充主要容器之前，制剂在超净工作台中使用0.2微米过滤器进行无菌过滤。填充也是无菌进行。
从SEC主峰数据可以看出，1号、4号处方的主峰下降较缓，2、3号处方的主峰降解较快；从IEC主峰数据可知，1号处方的主峰下降较缓，2、3、4号处方的主峰降解略快；从IEC酸区数据可知，4号处方的酸区增长较快，1、2、3号处方的酸区增长趋势一致。综合上述结果，最终选取1号处方，具体结果见表6。
表6a 100mg/ml制剂各处方SEC-HPLC和IEC-HPLC分析结果
Figure PCTCN2019074715-appb-000003
Figure PCTCN2019074715-appb-000004
表6b 80mg/ml制剂各处方SEC-HPLC和IEC-HPLC分析结果
Figure PCTCN2019074715-appb-000005
表6c 50mg/ml制剂各处方SEC-HPLC和IEC-HPLC分析结果
Figure PCTCN2019074715-appb-000006
Figure PCTCN2019074715-appb-000007
表6中的样品名称A161224-1/A161224-2/A161224-3/A161224-4分别对应表5中的序号1/2/3/4。
实施例3：稳定剂筛选研究
以下示出11个样品的实验及其结果。
11个样品中均含有：稳定剂(详见下表7)+缓冲液(5mM醋酸+5mM组氨酸缓冲液)+表面活性剂(0.1％的吐温80)+BAT1406抗体。抗体浓度分别为100mg/mL(高浓度)、80mg/mL(中浓度)、50mg/mL(低浓度)。
稳定剂筛选实验(高温40℃)：在高温40℃条件下，对11种稳定剂(见表7)在第0、5、10、15天时，进行SEC主峰、IEC主峰及酸区的分析。检测方法同实施例1。
表7稳定性成分
序号 样品名称 稳定剂 稳定剂浓度
1 A161209-1 -- --
2 A161209-2 山梨醇 4.20％
3 A161209-3 甘露醇 4.50％
4 A161209-4 氯化钠 0.90％
5 A161209-5 脯氨酸 2.50％
6 A161209-6 蔗糖 6％
7 A161209-7 海藻糖 8.4％
8 A161209-8 精氨酸盐酸盐 3.1％
9 A161209-9 甘氨酸 1％
10 A161209-10 甲硫氨酸 0.1％
11 A161209-11 氯化钙 10mM
对高、中、低三种抗体浓度的分析结果看，从SEC主峰数据作图可以看出，11组样品SEC主峰的变化趋势一致，其中1、4号处方的主峰下降幅度较大，其他组处方的主峰 降解趋势相差不大，结果见图4a-c；从IEC主峰数据作图可以看出，11组样品IEC主峰的变化趋势一致，其中4、8号处方的主峰下降幅度较小，其他组处方的主峰变化相差不大，结果见图5a-c；从IEC酸区数据作图可以看出，11组样品IEC酸区的变化趋势一致，其中4、8号处方的酸区增长幅度较小，其他组处方的酸区变化相差不大，结果见图5d-f。
稳定剂筛选实验(光照4000lx)：在光照4000lx条件下，对上述含有不同稳定剂的样品(见表7)在第0、5、10、15天时，进行SEC主峰、IEC主峰及酸区，CE-SDS(NR)的分析。检测方法见实施例。
对高、中、低三种抗体浓度的分析结果看，从SEC主峰数据作图可以看出，10号稳定剂的主峰变化最小，4号处方的主峰下降幅度最大，其他组稳定剂的主峰下降趋势相差不大，结果见图6a-c；从IEC主峰数据作图可以看出，10号稳定剂的主峰下降幅度最小，4、5号稳定剂的主峰下降较大，其他组稳定剂的主峰下降趋势一致结果见图7a-c；从IEC酸区数据作图可以看出，10号稳定剂的酸区增长最小，5、9号稳定剂的酸区增长较大，其他组稳定剂的酸区增长趋势一致结果见图7d-f；从CE-SDS(NR)主峰数据可以看出，10号处方的主峰下降最少，其他组处方的主峰下降趋势一致，具体结果见表8。
表8a稳定剂筛选CE-SDS(NR)分析结果(抗体浓度：100mg/ml)
Figure PCTCN2019074715-appb-000008
表8b稳定剂筛选CE-SDS(NR)分析结果(抗体浓度：80mg/ml)
Figure PCTCN2019074715-appb-000009
Figure PCTCN2019074715-appb-000010
表8c稳定剂筛选CE-SDS(NR)分析结果(抗体浓度：50mg/ml)
Figure PCTCN2019074715-appb-000011
综合上述SEC、IEC、CE-SDS(NR)在高温40℃及光照4000lx的分析结果看，高、中、低三种抗体浓度的变化趋势一致，4号(氯化钠)、5号(脯氨酸)、9号(甘氨酸)在高温及光照下均不稳定，其它组均较为稳定，较为突出的，可优先选用8号(精氨酸盐酸盐)、10号(甲硫氨酸)稳定剂，2号(山梨醇)和7号(海藻糖)。
实施例4：表面活性剂筛选
进行此试验，对比3种表面活性剂对抗体蛋白的保护作用，以确定表面活性剂浓度为多少时，能有效保护抗体的稳定性。
制备含不同浓度表面活性剂的蛋白溶液。具体配方见表9。溶液中的抗体蛋白浓度为100mg/mL、80mg/mL、50mg/mL，蛋白(BAT1406)不含表面活性剂，其他组分(5mM HAc，5mM His-HCl，3.1％Arg-HCl，0.1％Met，pH5.3)都含有。
表9不同配方蛋白溶液
序号 处方代码 成分
1 0.01％TW80 0.01％吐温80+BAT1406
2 0.1％TW80 0.1％吐温80+BAT1406
3 0.2％TW80 0.2％吐温80+BAT1406
4 0.01％TW20 0.01％吐温20+BAT1406
5 0.1％TW20 0.1％吐温20+BAT1406
6 0.2％TW20 0.2％吐温20+BAT1406
7 0.01％PX188 0.01％泊洛沙姆188+BAT1406
8 0.1％PX188 0.1％泊洛沙姆188+BAT1406
9 0.2％PX188 0.2％泊洛沙姆188+BAT1406
10 KB BAT1406(不含表面活性剂)
1、高温(40℃)试验
取上述10组样品配方，抗体蛋白(BAT1406)浓度分别为100mg/mL、80mg/mL、50mg/mL，分装，进行高温试验，对高温1周(1w)和高温2周(2w)后的样品进行SEC-HPLC检测，并与未高温(0D)的样品进行对比，检测结果如表10a-c：
表10a不同配方蛋白溶液(100mg/mL)高温后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000012
表10b不同配方蛋白溶液(80mg/mL)高温后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000013
表10c不同配方蛋白溶液(50mg/mL)高温后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000014
Figure PCTCN2019074715-appb-000015
从表10中数据可以看出，高温1周、2周后的含不同浓度BAT1406抗体的样品，其SEC-HPLC数据都相差不大，SEC-HPLC单体纯度降解趋势一致，不同表面活性剂之间的差异较小。
2、光照(4000lx，25℃)试验
制备如表9所述的10组样品配方，抗体蛋白(BAT1406)浓度分别为100mg/mL、80mg/mL、50mg/mL，分装，进行光照试验，对光照5天(5D)和光照10天(10D)后的样品进行SEC-HPLC检测，并与未光照(0D)的样品进行对比，检测结果如表11a-c：
表11a不同配方蛋白溶液(100mg/mL)光照后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000016
Figure PCTCN2019074715-appb-000017
表11b不同配方蛋白溶液(80mg/mL)光照后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000018
Figure PCTCN2019074715-appb-000019
表11c不同配方蛋白溶液(50mg/mL)光照后的SEC-HPLC检测结果
Figure PCTCN2019074715-appb-000020
Figure PCTCN2019074715-appb-000021
从表11中数据可以看出，光照5天、10天后的含不同浓度BAT1406抗体的样品，其SEC-HPLC数据相差不大，SEC-HPLC单体纯度降解趋势一致，不同表面活性剂之间的差异较小。
3、极限稀释
分别取相同体积含不同浓度表面活性剂的蛋白溶液，抗体蛋白(BAT1406)浓度分别为100mg/mL、80mg/mL、50mg/mL，按比例(100μL样品：20mL 0.9％氯化钠溶液)加入到0.9％NaCl溶液(生理盐水)中，轻微、充分混匀后观察其性状，并对放置2h后的蛋白溶液进行不溶性微粒检测。
不溶性微粒的检测方法为：依照《中华人民共和国药典2015年版第四部》通则0903：不溶性微粒检查法。仪器清洗至合格后，在洁净工作台中小心合并4个供试品内容物，置于取样杯中，静置2分钟脱气泡，置于取样器上。开启搅拌，使溶液混匀(避免气泡产生)，依法测定4次，每次进样量为4.0ml。弃第一次测定数据，取后续3次测定数据的平均值作为测定结果，根据取样体积与每个容器的标示装置体积，计算每个容器所含的微粒数。
检测结果见表12a-c，图8。
表12a 100mg/ml蛋白溶液在生理盐水中的微粒变化情况
Figure PCTCN2019074715-appb-000022
表12b 80mg/ml蛋白溶液在生理盐水中的微粒变化情况
Figure PCTCN2019074715-appb-000023
Figure PCTCN2019074715-appb-000024
表12c 50mg/ml蛋白溶液在生理盐水中的微粒变化情况
Figure PCTCN2019074715-appb-000025
对表12中微粒均值数据进行作图，得图8。从图8可以看出，不含表面活性剂的样品(KB组)的微粒数较其他组高很多，加有0.01％含量表面活性剂的样品的微粒数显著下降，当表面活性剂浓度升高到0.1％时，微粒数降到同生理盐水一致，继续增加到0.2％的浓度，样品微粒数变化不大，且表面活性剂的含量不宜太大。3种表面活性剂的差异也不大。
参照市售单抗制剂中表面活性剂的种类和含量，优选吐温80，含量定为0.1％。
实施例5：不同辅料处方对抗体稳定性的影响
处方研究-1(40℃高温稳定性实验)：具体组合成分及含量见表13(其中，抗体BAT1406含量分别为100mg/mL、80mg/mL、50mg/mL)。在温度40℃条件下，放置0天、 1周、3周、4周后，分析SEC主峰及IEC主峰、酸区的情况。
从SEC主峰数据作图可知(图9a-c)，1、7、8、9、12、14、15号样品的SEC单体纯度稳定性较好；从IEC主峰数据作图(图10a-c)可知，1、3、4、9、15号样品的IEC主峰含量稳定性较好；从IEC酸区数据作图(图10d-f)可知，1、2、3、4、10号样品的IEC酸区含量稳定性较好。
处方研究-2(4000lx光照稳定性实验)：具体组合成分及含量见表13(其中，抗体BAT1406含量分别为100mg/mL、80mg/mL、50mg/mL)。在光照4000lx条件下，分析SEC主峰以及IEC主峰、酸区在第0天，第7天，第14天时的情况进行分析。
从SEC主峰数据作图(图11a-c)可知，8、9、12、15号处方的SEC主峰含量下降较大；从IEC主峰数据作图(图12a-c)可知，5、6、8、9、14、15号处方的IEC主峰含量下降较大；从IEC酸区数据作图(图12d-f)可知，1、8、9、12、15号处方的IEC酸区含量增长较快。
表13不同辅料处方成分组成及含量
Figure PCTCN2019074715-appb-000026
Figure PCTCN2019074715-appb-000027
注：表13中的1-15号，分别对应图中序号A170112-1～A170112-15。
处方筛选研究-3(pH)：对3种稳定剂：精氨酸盐酸盐、海藻糖、山梨醇，(其中，抗体BAT1406含量分别为100mg/mL、80mg/mL、50mg/mL，筛选体系的其余组分见表14)。在pH 4.8～5.6的范围内，在温度40℃，放置0天、1周、3周、4周后，对SEC主峰、IEC主峰及酸区的情况进行分析；光照度4000lx的条件下，放置0天、1周、2周后，对SEC主峰、IEC主峰及酸区的情况进行分析，分析结果见图13-16。
温度40℃条件下，从SEC数据分析可知，3组稳定剂样品的SEC单体纯度都随样品pH的降低而降低，其中精氨酸盐酸盐(Arg-HCl)组样品受到pH的影响比较大，海藻糖(Tre)组样品受pH影响小。从整体水平上看，SEC单体纯度方面，海藻糖(Tre)优于山梨醇(Sor)优于精氨酸盐酸盐(Arg-HCl)。其中精氨酸盐酸盐(Arg-HCl)5.67组样品和精氨酸盐酸盐(Arg-HCl)5.44组样品的SEC单体纯度接近海藻糖(Tre)组和山梨醇(Sor)，精氨酸盐酸盐(Arg-HCl)4.82组样品、山梨醇(Sor)4.77组样品以及海藻糖(Tre)4.77组样品的SEC稳定性较差，故pH优选5.0～5.6，结果见图13a-c；从IEC主峰数据分析可知，3组稳定剂样品的IEC主峰含量都随样品pH的降低而降低，其中精氨酸盐酸盐(Arg-HCl)组样品受到pH的影响比较大，精氨酸盐酸盐(Arg-HCl)4.82组样品的稳定性较差，海藻糖(Tre)、山梨醇(Sor)组样品受pH影响小。从整体水平上看，IEC主峰含量方面，海藻糖(Tre)同山梨醇(Sor)相当，均优于精氨酸盐酸盐(Arg-HCl)。精氨酸盐酸盐(Arg-HCl)5.67和精氨酸盐酸盐(Arg-HCl)5.44组样品的IEC主峰含量明显优于海藻糖(Tre)和山梨醇(Sor)组，结果见图14a-c；从IEC酸区数据分析可知，3组稳定剂样品的IEC酸区含量都随样品pH的降低而降低，其中精氨酸盐酸盐(Arg-HCl)组样品受到pH的影响较大，海藻糖(Tre)、山梨醇(Sor)组样品受pH影响小。从整体水平上看，IEC酸区含量方面，精氨酸盐酸盐(Arg-HCl)优于海藻糖(Tre)和山梨醇(Sor)组，结果见图14d-f。综合SEC-HPLC和IEC-HPLC的结果，控制好pH，优选pH在5.0～5.6之间，样品的稳定性较好。
在光照4000lx的条件下，从SEC数据分析可知，3种稳定剂的表现看，精氨酸盐酸盐(Arg-HCl)组样品的SEC稳定性整体上优于山梨醇(Sor)组和海藻糖(Tre)组；且3组稳定剂在略低pH的条件下，稳定性较好，结果见图15a-c；从IEC主峰数据分析可知，3种稳定剂的表现看，精氨酸盐酸盐(Arg-HCl)组样品的IEC稳定性整体上优于山梨醇(Sor)组和海藻糖(Tre)组，结果见图16a-c；从IEC酸区数据分析可知，3种稳定剂的表现看，精氨酸盐酸盐(Arg-HCl)组样品的IEC稳定性整体上优于山梨醇(Sor)组和海藻糖(Tre)组；当pH＜5.4时，3组稳定剂在酸区含量上的表现相差不大，结果见图16d-f。
综上所述，高温(40℃)条件下，精氨酸盐酸盐(Arg-HCl)组样品受到pH的影响比较大，海藻糖(Tre)组样品受pH影响小。从整体水平上看，SEC单体纯度方面和IEC主峰含量方面，海藻糖(Tre)优于山梨醇(Sor)优于精氨酸盐酸盐(Arg-HCl)；IEC酸 区含量方面，精氨酸盐酸盐(Arg-HCl)优于海藻糖(Tre)和山梨醇(Sor)组。精氨酸盐酸盐(Arg-HCl)5.67组样品和精氨酸盐酸盐(Arg-HCl)5.44组样品的SEC单体纯度接近海藻糖(Tre)组和山梨醇(Sor)组，精氨酸盐酸盐(Arg-HCl)5.67和精氨酸盐酸盐(Arg-HCl)5.44组样品的IEC主峰含量明显优于海藻糖(Tre)和山梨醇(Sor)组。光照(4000lx)条件下，精氨酸盐酸盐(Arg-HCl)组样品的SEC单体纯度、IEC主峰含量稳定性整体上优于山梨醇(Sor)组和海藻糖(Tre)组；且3组稳定剂在略低pH的条件下，稳定性较好。当pH＜5.4时，3组稳定剂在酸区含量上的表现相差不大，由此可筛选出3组优选制剂处方组合，具体看表14a；更优选的制剂处方组合见表14b。
其中，综合稳定性最为突出的为精氨酸盐酸盐(Arg-HCl)，尤其是精氨酸盐酸盐(Arg-HCl)5.44组。目前，市面上的单抗制剂的稳定剂多采用多元醇和糖类，我们的实验结果显示，高温(40℃)条件下，精氨酸盐酸盐(Arg-HCl)组样品的IEC-HPLC酸区含量低于多元醇和糖类，表明此处方在减缓样品酸区增长方面更具优势；此外，光照(4000lx)条件下，精氨酸盐酸盐(Arg-HCl)组样品的SEC单体纯度、IEC主峰含量稳定性整体上也优于多元醇和糖类。
注：在本实施例中精氨酸盐酸盐组指制剂中的稳定剂为3.1％精氨酸盐酸盐+0.1％甲硫氨酸。
表14a优选制剂处方组合
Figure PCTCN2019074715-appb-000028
表14b更优选的制剂处方组合
Figure PCTCN2019074715-appb-000029
实施例6：生物活性研究
取对数生长期的L-929，接种于96孔板中，8000cells/孔/100ul。第2天，用含10％FBS和rhTNF-α(浓度1ng/ml)的培养基稀释1406抗体、hIgG阴性对照组(起始浓度为750ng/ml，以1/3稀释浓度往下稀释，每个浓度设2个复孔，共设立8个梯度)。混匀，室温下共孵育30分钟后，再加入5000ng/ml的放线菌素D，使其浓度达到200ng/ml。混匀，把上述混合物加到L-929细胞中，100ul/孔(终体积为200ul/孔，抗体、rhTNF-α和放线菌素D对应终浓度减半)，37℃/5％CO 2培养箱中培养24小时。24小时后，将板中的培养基甩掉，加入新鲜培养基100ul/孔，加入10ul/孔的CCK-8，37℃、5％CO 2培养箱中继续培养4小时，OD450读数。计算1406抗体的EC50。
室温稳定性研究：将BAT1406抗体制剂(A1、B1、C1、A2、B2、C2、A3、B3、C3制剂，具体组分见表1)保存在室温下，进行体外TNF中和试验(L-929生物活性检测)，稳定性研究时间为4周。体外中和TNF试验结果显示，制剂A1、B1、C1、A2、B2、C2、A3、B3、C3九种制剂在第0天时，EC50分别为19.395ng/ml、20.489ng/ml、20.565ng/ml、21.806ng/ml、21.856ng/ml、19.722ng/ml、23.585ng/ml、21.448ng/ml、19.922ng/ml，在室温放置4周后，EC50分别为20.465ng/ml、20.385ng/ml、20.921ng/ml、20.817ng/ml、21.812ng/ml、20.934ng/ml、20.227ng/ml、20.583ng/ml、20.33ng/ml，其生物学活性未受影响，具体结果见图17a-i。
长期稳定性研究(2～8℃)：将BAT1406抗体制剂(A1、B1、C1、A2、B2、C2、A3、B3、C3制剂)保存在2～8℃下，稳定性研究时间为36个月。分别在第3个月，第6个月，第12个月，第18个月，第24个月，第30个月，第36个月时，取样品进行体外TNF中和试验(L-929生物活性检测)，结果显示，在第0个月，第3个月，第6个月，第12个月，第18个月，第24个月，第30个月，第36个月时，生物活性基本不变，表明A1、B1、C1、A2、B2、C2、A3、B3、C3制剂在2～8℃下至少能保持36个月的稳定，具体结果见表15。
表15a BAT1406(100mg/ml)三种制剂的2～8℃条件下36个月稳定性考察
Figure PCTCN2019074715-appb-000030
Figure PCTCN2019074715-appb-000031
表15b BAT1406三种制剂(80mg/ml)的2～8℃条件下36个月稳定性考察
Figure PCTCN2019074715-appb-000032
表15c BAT1406三种制剂(50mg/ml)的2～8℃条件下36个月稳定性考察
Figure PCTCN2019074715-appb-000033
Figure PCTCN2019074715-appb-000034
实施例7：制剂反复冻融研究
冻融研究-1:将BAT1406抗体制剂(A、B、C制剂)分别配制成3种浓度，低浓度样品LOQ(50mg/mL)、中浓度样品MOQ(80mg/mL)、高浓度样品HOQ(100mg/mL)各3个样本(具体组分见表1)，从-80℃冰箱中取出，室温放置大于2h，融化后再冻存，如此循环5次，两次融化间的冻存时间不少于12h。复孔测定冻融后的质控的浓度值，比较测定值和理论值，考察5次冻融后样品的稳定性。每个样品分别稀释10万倍进行Elisa检测，复孔测定的回收率和精密度列于表16。
表16重组人抗TNF-α单克隆抗体BAT1406三种制剂的冻融实验结果
Figure PCTCN2019074715-appb-000035
结果表明，BAT1406抗体制剂(A制剂)三个浓度的样品5次冻融后，其回收率范围为99.6～109.4％，精密度范围为0.8～4.8％，BAT1406抗体制剂(B制剂)三个浓度的样品5次冻融后，其回收率范围为100.2～110.7％，精密度范围为0.6～6.7％，BAT1406抗体制剂(C制剂)三个浓度的样品5次冻融后，其回收率范围为98.4～107.9％，精密度范围为0.5～5.8％，说明抗体制剂在冻存条件下，无论是低、中、高浓度，在反复冻融5次情况下，其稳定性均满足实验要求。值得注意的是，所述抗体在高浓度100mg/mL，A、B、C三种制剂反复冻融5次后回收率仍可高达98％以上，这是非显而易见的，因为抗体制剂一般在高浓度下更不稳定，高浓度的抗体制剂可能会伴随抗体聚集、可溶性降低、稳定性下降等负面作用。
冻融研究-2:将药物制剂(A1、B1、C1、A2、B2、C2、A3、B3、C3制剂)从冷冻状态转变成液体状态循环5次，评估蛋白浓度为100mg/ml、80mg/ml、50mg/ml的BAT1406抗体药物的冻融表现。制剂(A1、B1、C1、A2、B2、C2、A3、B3、C3制剂)分别从-80℃或-20℃开始评估5次快速和慢速冷/融循环的效果的实验结果见表17。结果表明BAT1406抗体药物制剂可以解冻/冷冻至少5次，而对物理化学特性或者生物活性不产生任何有害的作用。结果还表明了无论是快慢速或快速冻/融循环，都具有较少数量的只有在显微镜下才能看到的颗粒；体外中和TNF试验亦表明其生物学活性未受影响。
表17a重组人抗TNF-α单克隆抗体BAT1406(100mg/mL)的制剂冻/融实验结果
Figure PCTCN2019074715-appb-000036
Figure PCTCN2019074715-appb-000037
表17b重组人抗TNF-α单克隆抗体BAT1406(80mg/mL)的制剂冻/融实验结果
Figure PCTCN2019074715-appb-000038
表17c重组人抗TNF-α单克隆抗体BAT1406(50mg/mL)的制剂冻/融实验结果
Figure PCTCN2019074715-appb-000039
Figure PCTCN2019074715-appb-000040
实施例8：微生物研究
需要对药物制剂(A1、B1、C1、A2、B2、C2、A3、B3、C3制剂)进行微生物研究，以便确定所述制剂是否能支持微生物生长。通过用微生物(例如，金黄色葡萄球菌，ATDD-NO.:6538p，白色念球菌，ATDD-NO.:10231，黑曲霉，ATDD-NO.:16404，环境分离物)在低水平(NMT100cfu/ml)下直接接种所述无菌制剂，然后检查接种过的制剂的总体微生物生长。评价的指标主要有显微镜下微生物的数目以及浊度的变化，其中，浊度的缺乏是没有总体生长的指标，并且在14天之后在接种的容器中检测。另外，从这些容器中不能重新分离到微生物。表18表明，如果在室温20-25℃保存14天，所述制剂不支持微生物生长。
表18a重组人抗TNF-α单克隆抗体BAT1406(100mg/mL)三种制剂的微生物检测
Figure PCTCN2019074715-appb-000041
表18b重组人抗TNF-α单克隆抗体BAT1406(80mg/mL)三种制剂的微生物检测
Figure PCTCN2019074715-appb-000042
Figure PCTCN2019074715-appb-000043
表18c重组人抗TNF-α单克隆抗体BAT1406(50mg mg/mL)三种制剂的微生物检测
Figure PCTCN2019074715-appb-000044
-：浊度不变
实施例9：皮下注射体内代谢情况
分别对SD大鼠进行A1、B1、C1、A2、B2、C2、A3、B3、C3制剂的皮下注射，比较三种制剂在SD大鼠体内的药物代谢情况。首先，称量动物体重(平均重量100g/只)，按32mg/kg计算每只大鼠所需要的抗体量，每只大鼠注射量约为3.2mg抗体(每组给药方式3只SD大鼠，合共27只大鼠)。注射抗体前，先取SD大鼠0h的血清，-80℃冻存备用。注射抗体后，分别在1h、4h、24h、48h、72h、96h、7day、9day、11day、13day、15day、22day、29day、36day、54day分别剪SD大鼠尾部取血样，分离血清，-80℃冻存备用。然后通过ELISA检测血液中抗体浓度，对组内数据进行平均并分析，从而评价药物在不同制剂条件下使用的代谢过程。结果表明(见图18a-c)重组人抗TNF-α单克隆抗体BAT1406蛋白在九种制剂条件下，对SD大鼠进行皮下注射，BAT1406蛋白的药物代谢情况基本保持一致，半衰期约为14天，满足临床要求。证明九种制剂可以用于进行皮下注射。
实施例10：重组人抗TNF-α单克隆抗体BAT1406蛋白序列
一种治疗TNF-α相关疾病的人抗体为重组人抗TNF-α单克隆抗体BAT1406，是通过基因工程手段，在CHO细胞中表达的，并且通过一系列标准的层析步骤纯化获得的。BAT1406属于IgG抗体，分子量为148kDa，由2条IgG1 z,a重链和2条κ轻链组成。每条重链含有451个氨基酸，分子量为49kDa，其重链氨基酸序列如SEQ ID NO.1；每条轻链含有214个氨基酸，分子量为24kDa，轻链氨基酸序列如SEQ ID NO.2。重组人抗TNF-α单克隆抗体BAT1406重链氨基酸序列
SEQ ID NO.1
Figure PCTCN2019074715-appb-000045
Figure PCTCN2019074715-appb-000046
重组人抗TNF-α单克隆抗体BAT1406轻链氨基酸序列
SEQ ID NO.2
Figure PCTCN2019074715-appb-000047
实施例11：重组人抗TNF-α单克隆抗体BAT1406的表达和纯化
参照Wood et al.,J Immunol.145:3011(1990)等的方法，特异性结合TNF-α的单克隆抗体抗TNF-α抗体在CHO细胞表达。含抗体基因的表达载体用常规的分子生物学方法构建(Molecular Cloning)，以CHO-k1细胞(ATCC CCL61)的一种衍生细胞系作为宿主细胞表达。高产稳定细胞系的构建过程简单描述如下：宿主细胞悬浮生长于CD-CHO培养基(Gibco,CA)，取处于对数生长期的宿主细胞离心，重悬于新鲜的CD-CHO培养基，计数并调节细胞密度到1.43×10 7个/毫升，取600ul上述细胞悬液加入电击杯，然后加入已线性化的质粒40ug，(用移液枪吸打使细胞与质粒混合均匀。用Bio-rad电转仪电击转化，仪器参数设定为：电容：960uFD,电压：300V。通常电击时间为15-20毫秒为正常。把电击后的细胞立即重悬于37℃预热的CD-CHO培养基)，每孔100ul分装于96孔板，2-3天后加入等量的筛选培养基(CD-CHO media+50uM MSX)。测定96孔板细胞培养上清来测定抗体的表达水平。表达水平较高的克隆从96孔板转移到24孔板，待细胞生长到一定数量，把细胞转入6孔板，使每孔5ml培养基含2×10 5个细胞，测定细胞的抗体产量和产率。通常20-30个克隆被转到摇瓶做进一步评价。最后选取5-8个表达量最高的克隆进行亚克隆和进一步的表达检测。收获料液，通过低速离心使细胞和培养基分离，把离心上清高速离心进一步澄清。用蛋白A亲和纯化和离子交换纯化，使用的介质分别是GE公司生产的Mab Select SuRe和Capto S。
实施例12：重组抗体BAT1406的特异性研究
药理学研究主要进行了重组人抗TNF-α单克隆抗体在体外药效试验。体外试验主要进行下面几个方面的药效学检测：与TNF的结合能力，抗体特异性、对TNF与受体结合的竞争性，对TNF-α生物学活性的抑制作用以及细胞毒实验等。以抗体特异性结合为例，比较重组人抗TNF-α单克隆抗体与TNF-α的特异性结合能力。具体实验方法如下：首先用PBS将rhTNF-α、rhTNF-β或者rmTNF-α稀释成100ng/100μl，将稀释好的rhTNF-α、rhTNF-β或者rmTNF-α加到96孔酶标板中，100μl/孔，4℃过夜。次日用含3％BSA的PBS封闭，200μl/孔，置37℃2小时。弃上清后，往对应孔中分别加入不同浓度的重组人抗TNF-α单抗BAT1406和人IgG(抗体终浓度为60.75ug/ml，3倍稀释，10个梯度，复 孔)，37℃下作用2小时。弃上清后，PBST洗板5次，拍干。然后以1：10000加入羊抗人HRP二抗，100μl/孔，37℃1小时。弃上清后，PBST洗板6次，加入TMB显色液，100μl/孔。OD450读数，作标准曲线。结果表明不同浓度的重组人抗TNFα单抗BAT1406对rhTNF-α有特异性结合并呈现浓度梯度结果，结果保持一致；但是不同浓度的重组人抗TNF-α单抗BAT1406对rhTNF-β或者rmTNF-α没有特异结合。阴性对照人IgG对rhTNF-α、rhTNF-β或者rmTNF-α都没有特异结合，证明了BAT1406只与rhTNF-α结合(IC50＝3×10 - 9M)，而不与rhTNF-β和rmTNF-α结合。
参考文献：
[1]Marc Feldmann,Ravinder N.Maini.Discovery of TNF-αas a therapeutic target in rheumatoid arthritis preclinical and clinical studies.Joint Bone Spine,2009；69:12-18.
[2]Feldmann M,Brennan FM,Foxwell BMJ,et al.The role of TNF and IL-1in rheumatoid arthritis.Curr Dir Au toimmun,2001；3:188-199.
[3]Daniel Tracey,Lars Klareskog,Eric H.Sssso,et al.Tumor necrosis factor antagonist mechanisms of action A comprehensive review.PHarmacology and Therapeutics,2008；117:244-279.
[4]D.J.Shealy,S.Visvanathan.Anti-TNF Antibodies:Lessons from the Past,Roadmap for the Future.Handbook of Experimental PHarmacology 2008；181:101-129.
[5]B.Gatto.Biologics targeted at TNF:design,production and challenges.Reumatismo,2006；58(2)：94-103.

Claims (11)

  1. 一种抗体制剂，其特征在于含有以下组分：
    (1)20～120mg/ml的抗TNF-α人源抗体
    (2)缓冲剂
    (3)0.5-10％稳定剂
    (4)0.01％～0.2％表面活性剂
    (5)注射用水
    所述制剂的pH为4.0～7.0；
    优选地，所述抗体的浓度为40～120mg/ml，更优选为50～100mg/ml；
    优选地，所述的缓冲剂选自琥珀酸、柠檬酸、His-HCl、醋酸、磷酸盐中的一种或多种；更优选地，所述的缓冲剂选自His-HCl、HAc、磷酸盐和HAc的组合、His-HCl和HAc的组合；更优选His-HCl和HAc的组合；更优选地，每种缓冲剂浓度均为5～15mM；更优选5～10mM；
    更优选地，当所述缓冲剂选自两种的组合时，前者与后者在体系中的摩尔比为1:2～2:1；更优选1:1；
    优选地，所述的稳定剂选自Arg-HCl和Met的组合，或者山梨醇，或者海藻糖；
    优选地，所述的表面活性剂选自吐温80、吐温20或泊洛沙姆188。
  2. 根据权利要求1所述的制剂，其特征在于：所述的重组人抗TNF-α单克隆抗体具有SEQ ID NO.1所示的重链以及SEQ ID NO.2所示的轻链。
  3. 根据权利要求1所述的制剂，其特征在于：
    所述的稳定剂为0.5％～5.0％Arg-HCl和0.01％～2％Met；
    或者优选地，所述的稳定剂选自3.0％～6.0％山梨醇；
    又或者优选地，所述的稳定剂选自7％～10％海藻糖。
  4. 根据权利要求3所述的制剂，其特征在于含有以下组分：
    (1)45～120mg/ml的抗TNF-ɑ人源抗体
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)2％～3.5％Arg-HCl
    (5)0.01％～0.5％Met
    (6)0.01％～0.1％Tween 80
    (7)注射用水
    其中，所述制剂的pH为5.0～5.6，优选地，pH为5.2～5.6；
    或者优选地，含有以下组分：
    (1)45～120mg/ml的抗TNF-ɑ人源抗体
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)4.0％～5.0％山梨醇
    (5)0.01％～0.1％Tween 80
    (6)注射用水
    其中，所述制剂pH为5.0～5.6，优选地，pH为5.0～5.4；
    又或者优选地，含有以下组分：
    (1)45～120mg/ml有效量的抗TNF-ɑ人源抗体
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)7％～9％海藻糖
    (5)0.01％～0.1％Tween 80
    (6)注射用水
    所述的制剂的pH为5.0～5.6，优选地，pH为5.0～5.4。
  5. 根据权利要求3所述的制剂，其特征在于含有以下组分：
    (1)80～120mg/ml的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)2％～3.5％Arg-HCl
    (5)0.01％～0.5％Met
    (6)0.01％～0.1％Tween 80
    (7)注射用水
    其中，所述制剂的pH为5.0～5.6，优选地，pH为5.2～5.6；
    或者优选地，含有以下组分：
    (1)80～120mg/ml的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)4.0％～5.0％山梨醇
    (5)0.01％～0.1％Tween 80
    (6)注射用水
    其中，所述制剂的pH为5.0～5.6，优选地，pH为5.0～5.4；
    又或者优选地，含有以下组分：
    (1)80～120mg/ml有效量的抗TNF-ɑ人源抗体；优选85～120mg/ml；更优选90～110mg/ml
    (2)5～10mM HAc
    (3)5～10mM His-HCl
    (4)7％～9％海藻糖
    (5)0.01％～0.1％Tween 80
    (6)注射用水
    所述的制剂的pH为5.0～5.6，优选地，pH为5.0～5.4。
  6. 根据权利要求4所述的制剂，其特征在于含有以下组分：
    (1)100mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)3.1％Arg-HCl
    (5)0.1％Met
    (6)0.1％Tween 80
    (7)注射用水
    其中，所述制剂的pH为5.3±0.3，优选地，pH为5.4±0.2；
    或者优选地，含有以下组分：
    (1)50mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)3.1％Arg-HCl
    (5)0.1％Met
    (6)0.1％Tween 80
    (7)注射用水
    其中，所述制剂的pH为5.3±0.3，优选地，pH为5.4±0.2；
    或者优选地，含有以下组分：
    (1)100mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)4.2％山梨醇
    (5)0.1％Tween 80
    (6)注射用水
    其中，所述制剂的pH为5.3±0.3，优选地，pH为5.2±0.2；
    或者优选地，含有以下组分：
    (1)50mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)4.2％山梨醇
    (5)0.1％Tween 80
    (6)注射用水
    其中，所述制剂的pH为5.3±0.3，优选地，pH为5.2±0.2；
    又或者优选地，含有以下组分：
    (1)100mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)8.4％海藻糖
    (5)0.1％Tween 80
    (6)注射用水
    所述的制剂的pH为5.3±0.3，优选地，pH为5.2±0.2。
    又或者优选地，含有以下组分：
    (1)50mg/ml的抗TNF-ɑ人源抗体
    (2)5mM HAc
    (3)5mM His-HCl
    (4)8.4％海藻糖
    (5)0.1％Tween 80
    (6)注射用水
    所述的制剂的pH为5.3±0.3，优选地，pH为5.2±0.2。
  7. 根据权利要求1-6任一所述的制剂，其特征在于：所述制剂的渗透压为150～400mOsm/Kg；优选地，渗透压为260～340mOsm/Kg；
    优选地，所述的制剂还含有NaOH用于调节pH。
  8. 根据权利要求1-6任一所述的制剂，其特征在于：所述制剂为注射制剂；优选地，所述制剂为皮下注射制剂。
  9. 根据权利要求1-6任一所述的制剂，其特征在于：所述制剂在室温下保持稳定至少1个月；优选地，该制剂在2-8℃下至少能稳定36个月；优选地，该制剂至少5次循环冻融后保持稳定性。
  10. 根据权利要求1-6任一所述的制剂，其特征在于，所述制剂为治疗由TNF-α引起的病症的药物制剂；优选地，所述的由TNF-α引起的病症包含脓毒症、自身免疫性疾病、传染病、移植、恶性肿瘤、肺病、肠道疾病、心脏病、颈椎关节病或皮肤病；更优选地，所述的由TNF-α引起的病症包含类风湿关节炎、牛皮癣性关节炎或强直性脊柱炎。
  11. 制备权利要求1-10任一所述的抗体制剂的方法，其特征在于包含以下步骤：
    (1)配置包含稳定剂的缓冲体系；
    (2)通过UF/DF超滤，采用步骤(1)制备的缓冲液对抗体进行超滤换液，然后进行浓缩至抗体的目标浓度；
    (3)添加表面活性剂到步骤(2)制备的液体中；
    (4)过滤除菌后填充到容器。
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