High-concentration monoclonal antibody preparation and application thereof

Abstract

According to the present invention, a high concentration antibody preparation is provided, the antibody preparation comprises a monoclonal antibody and a surfactant, the surfactant is polysorbate and poloxamer, and the concentration of the monoclonal antibody is 100 mg / ml to 200 mg / ml. The high-concentration antibody preparation provided by the invention has strong stability under freeze thawing, oscillation, acceleration and long-term conditions, can maintain good stability in preparation, transportation and storage processes, and ensures clinical medication safety and quality controllability.

Description

A high concentration monoclonal antibody preparation and its application

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a high-concentration monoclonal antibody preparation and application thereof.

[0002] As a protein drug, antibodies are affected by a variety of environmental factors during storage compared to conventional chemical drugs, such as being highly sensitive to temperature, oxygen, and ultraviolet rays. Due to the effects of these factors, a variety of physical or chemical changes may occur. Physical changes include degradation and aggregation (soluble and insoluble aggregates); chemical changes include hydrolysis and oxidation. These changes will cause the stability of antibodies to be unable to be guaranteed during storage, resulting in the following effects: on the one hand, the efficacy is affected by changes in the actual dosage, and on the other hand, side effects may occur due to the production of related substances.

[0003] Especially for high-concentration antibody preparations, their stability is even more difficult to maintain. Nonionic surfactants are widely used in the production, transportation, freezing and thawing, and long-term storage of biological preparations to prevent protein aggregation and particle formation. Among them, polysorbate 20 and polysorbate 80 are the most commonly used nonionic surfactants for biological preparations, but studies have found that the chemical properties of polysorbate 20 and 80 (PS20, PS80) are unstable and often oxidize and hydrolyze. Polysorbate hydrolysis will form visible or invisible particles in the preparation, making the drug preparation unsuitable for injection; polysorbate oxidation will reduce its concentration and fail to achieve the expected effect, and will also produce peroxides, aldehydes, organic acids and other harmful substances, which will also cause changes in proteins. Poloxamer 188 (P188) is the only surfactant used in marketed injectable biological preparations other than polysorbate. In addition to competing with protein molecules in the preparation to bind to the hydrophobic layer, it can also directly bind to protein molecules to prevent protein aggregation and precipitation.

[0004] There are more than 100 kinds of antibody drugs currently on the market, among which there are dozens of high-concentration antibody (>140mg / mL) drugs, but all of them use a single surfactant. The applicant of the present invention has found through research that due to the special properties of high-concentration antibodies, a large number of particles visible to the naked eye will be formed in a very short time under oscillation conditions when only the surfactant polysorbate is used. Although poloxamer can effectively control the formation of aggregation for a certain period of time, it still cannot protect the stability of antibodies well in many aspects. The present invention uses a double surfactant (polysorbate and poloxamer) to protect the target antibody. Compared with a single surfactant, the formulation containing a double surfactant can better keep the high-concentration antibody in good stability.

[0005]

[0006] The present invention discloses a high-concentration antibody preparation, which comprises a monoclonal antibody and a surfactant, wherein the surfactant is polysorbate and poloxamer, and the concentration of the monoclonal antibody is 100 mg / ml-200 mg / ml, preferably 135 mg / ml-165 mg / ml; in some specific embodiments of the present invention, the antibody concentration is 100 mg / ml, 120 mg / ml, 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 180 mg / ml, 190 mg / ml or 200 mg / ml.

[0007] In some embodiments of the present invention, the polysorbate is polysorbate 20 and / or polysorbate 80; preferably, the poloxamer is poloxamer 188.

[0008] In some embodiments of the present invention, the concentration of the polysorbate is 0.02 mg / ml-0.45 mg / ml, preferably 0.08 mg / ml-0.4 mg / ml, and more preferably 0.1 mg / ml-0.3 mg / ml; in some specific embodiments of the present invention, the concentration of the polysorbate is 0.02 mg / ml, 0.03 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.10 mg / ml, 0.12 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml, 0.35 mg / ml, 0.4 mg / ml or 0.45 mg / ml.

[0009] In some embodiments of the present invention, the concentration of the poloxamer is 0.4 mg / ml-1.5 mg / ml, preferably 0.8 mg / ml-1.2 mg / ml, and more preferably 1.0 mg / ml; in some specific embodiments of the present invention, the concentration of the poloxamer is 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml or 1.5 mg / ml.

[0010] In some embodiments of the present invention, the antibody preparation further comprises a stabilizer. Preferably, the stabilizer is any one or more of sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine, glycine, proline, and lysine; more preferably, the stabilizer is any one or more of sucrose, trehalose, sodium chloride, or arginine.

[0011] In some embodiments of the present invention, the stabilizer is: sucrose, trehalose, maltose, sorbitol and / or mannitol at a concentration of 40 mg / ml-80 mg / ml, preferably 50 mg / ml-80 mg / ml, more preferably 50 mg / ml-70 mg / ml, or sodium chloride, arginine, glycine, proline or lysine at a concentration of 100 mM-180 mM, more preferably 130 mM-150 mM; in the present invention, In some specific embodiments, the stabilizer is sucrose, trehalose, maltose, sorbitol and / or mannitol at a concentration of 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml or 80 mg / ml, or the stabilizer is 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, or 180 mM sodium chloride, arginine, glycine, proline or lysine.

[0012] In some embodiments of the present invention, the antibody preparation further comprises a buffer, preferably, the buffer is a citrate buffer, an acetate buffer and / or a histidine hydrochloride buffer.

[0013] In some embodiments of the present invention, the concentration of the buffer is 3mM-50mM; preferably, the concentration of the buffer is 10mM-30mM; in some specific embodiments of the present invention, the concentration of the buffer is 3mM, 5mM, 8mM, 10mM, 15mM, 20mM, 25mM or 30mM.

[0014] In some embodiments of the present invention, the pH value of the preparation is 4.8-5.5, preferably 5.0-5.4; in some specific examples of the present invention, the pH value of the preparation is 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

[0015] In some embodiments of the present invention, the antibody concentration is 100 mg / ml-200 mg / ml; the stabilizer is 40 mg / ml-80 mg / ml sucrose and / or trehalose, or the stabilizer is 100 mM-180 mM sodium chloride or arginine; the buffer is 3 mM-50 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 4.8-5.5; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer.

[0016] In some embodiments of the present invention, the antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-80 mg / ml of sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 4.8-5.5; the surfactant is 0.02 mg / ml-0.45 mg / ml of polysorbate and 0.4 mg / ml-1.5 mg / ml of poloxamer.

[0017] In some embodiments of the present invention, the antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer;

[0018] In some embodiments of the present invention, the antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose; the buffer is 10 mM-30 mM acetate buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer.

[0019] In some embodiments of the present invention, the preparation is any one of the following antibody preparations:

[0020] (1) The antibody concentration is 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml or 165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer;

[0021] (2) The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml, 60 mg / ml or 70 mg / ml of sucrose and / or trehalose, or the stabilizer is 130 mM, 140 mM or 150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml of polysorbate and 0.4 mg / ml-1.5 mg / ml of poloxamer;

[0022] (3) The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM, 20 mM or 30 mM acetate buffer, citrate buffer or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer;

[0023] (4) The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0, 5.1, 5.2, 5.3 or 5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer;

[0024] (5) The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.08 mg / ml, 0.10 mg / ml, 0.12 mg / ml, 0.15 mg / ml, 0.20 mg / ml, 0.30 mg / ml, 0.40 mg / ml or 0.45 mg / ml of polysorbate and 0.4 mg / ml-1.5 mg / ml of poloxamer;

[0025] (6) The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml or 1.5 mg / ml poloxamer;

[0026] The polysorbate is polysorbate 80 or polysorbate 20.

[0027] In some embodiments of the present invention, the antibody preparation is any one of the following preparations:

[0028] (1) The antibody concentration is 150 mg / ml; the stabilizer is 130 mM sodium chloride; the buffer is 20 mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0029] (2) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0030] (3) The antibody concentration is 135 mg / ml; the stabilizer is 50 mg / ml sucrose; the buffer is 10 mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0031] (4) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM acetate buffer; the pH value of the preparation is 5.2; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0032] (5) The antibody concentration is 165 mg / ml; the stabilizer is 70 mg / ml sucrose; the buffer is 30 mM acetate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0033] (6) The antibody concentration is 150 mg / ml; the stabilizer is 80 mg / ml sucrose; the buffer is 30 mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0034] (7) The antibody concentration is 150 mg / ml; the stabilizer is 150 mM arginine; the buffer is 20 mM acetate buffer; the pH value of the preparation is 5.2; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0035] (8) The antibody concentration is 160 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0036] (9) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 4.8; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0037] (10) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM histidine hydrochloride buffer; the pH value of the preparation is 5.5; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer;

[0038] (11) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.02 mg / ml or 0.05 mg / ml polysorbate and 1.5 mg / ml poloxamer;

[0039] (12) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.15 mg / ml polysorbate and 0.5 mg / ml poloxamer;

[0040] (13) The antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.45 mg / ml polysorbate and 0.4 mg / ml poloxamer; the polysorbate is polysorbate 80 or polysorbate 20.

[0041] In some embodiments of the present invention, the antibody preparation further comprises an antioxidant and / or a preservative. The preservative or antioxidant is a preservative or antioxidant commonly used in antibody preparations. The concentration of the oxidant and / or preservative is 0-20mM, and in some specific embodiments, the concentration of the antioxidant is 0, 5mM, 8mM, 10mM, 12mM, 15mM, 18mM or 20mM. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or a combination thereof; the antioxidant is methionine.

[0042] In some embodiments of the present invention, the antibody preparation is a liquid preparation or a powder for injection.

[0043] In some embodiments of the present invention, the antibody is an anti-thymic stromal lymphopoietin (TSLP) antibody or an anti-neonatal Fc receptor (FcRn) antibody. On the other hand, the present invention also provides the use of the above antibody preparation in the preparation of a drug for treating a disease; preferably, the disease includes an inflammatory or autoimmune disease.

[0044] On the other hand, the antibody preparations provided by the present invention are suitable for various administration methods known in the art, including but not limited to: intravenous injection, intraarterial administration, intraperitoneal injection, intrapulmonary administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration or transdermal administration. In some embodiments, the preparation is administered subcutaneously. In some embodiments, the preparation is administered intramuscularly.

[0045] The "antibodies" of the present invention include full-length antibodies and antigen-binding fragments thereof. Full-length antibodies include two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually include three highly variable loops, known as complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by Kabat, Chothia or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conservative than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of the full-length antibodies described in the present invention are IgA, IgD, IgE, IgG and IgM, characterized by having α, δ, ε, γ and μ heavy chains, respectively. Further, the several major antibody classes described in the present invention can be further divided into subclasses, including: IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain) or IgA2 (α2 heavy chain).

[0046] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibodies (dsdiabodies), single-chain antibodies (scFv), scFv dimers (divalent double-chain antibodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure.

[0047] The high-concentration antibody preparation provided by the present invention has the following excellent effects:

[0048] The antibody preparation provided by the present invention adopts a combination of two surfactants, polysorbate and poloxamer, which can more effectively prevent collision and contact between antibody molecules and reduce the formation of aggregates at the interface compared to the use of a single surfactant. The high-concentration antibody preparation of the present invention maintains strong stability under freeze-thaw, oscillation, acceleration and long-term conditions, and can maintain good stability during preparation, transportation and storage, ensuring clinical drug safety and quality controllability.

[0049] In order to make the technical problems to be solved, the technical solutions and advantages adopted by the present invention clearer, the present invention will be described in detail below in conjunction with specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0050] Unless otherwise specified, the reagents used in the following examples were prepared by conventional methods or obtained from commercial sources; the experimental methods used were conventional methods unless otherwise specified; the materials, instruments, etc. used were obtained from commercial sources unless otherwise specified.

[0051] The exemplary antibodies used in the embodiments of the present invention are TSLP-01-IgG1, TSLP-02-IgG1, FcRn-01-IgG4, FcRn-02-IgG4, FcRn-03-IgG4, and FcRn-04-IgG4, and their amino acid sequences are shown in Table 1:

[0052] Table 1. Amino acid sequences of antibodies

[0053] Example 1. Antibody preparation and screening

[0054] 1. Preparation and activity detection of anti-TSLP antibodies

[0055] (1) Preparation of anti-TSLP antibodies

[0056] scFv antibodies targeting TSLP targets were screened from the scFv antibody yeast display library. After further optimization, full-length anti-TSLP-01-IgG1 and TSLP-02-IgG1 antibodies were constructed and screened, and their amino acid sequences are shown in Table 1.

[0057] (2) TSLP-induced Stat5 activation inhibition experiment

[0058] The efficacy of the antibodies TSLP-01-IgG1 and TSLP-02-IgG1 shown in Table 1 in inhibiting human TSLP-stimulated signal transducer and activator of transcription 5 activation in Ba / F3 stable cells was evaluated and a cell-based assay was employed. As shown in Table 2, the TSLP-01-IgG1 antibody and the TSLP-02-IgG1 antibody showed better effects in inhibiting human TSLP-induced Stat5 activation than the reference antibody AMG157.

[0059] (3) TSLP-induced Ba / F3 cell proliferation inhibition experiment

[0060] The ability of anti-TSLP antibodies to inhibit TSLP-induced proliferation of Ba / F3 cells expressing human TSLP receptor (hTSLPR) and human IL-7Ra (hIL7Ra) was evaluated. As shown in Table 2, anti-TSLP antibodies showed better effects in inhibiting TSLP-induced proliferation of Ba / F3 cells compared with reference antibody AMG157.

[0061] (4) TARC release inhibition experiment in human PBMCs

[0062] To determine whether anti-TSLP antibodies can neutralize TSLP in primary cell-driven reactions, human TSLP-induced TARC secretion from human PBMCs was tested in the presence or absence of anti-TSLP antibodies. As shown in Table 2, TSLP-01-IgG1 antibody and TSLP-02-IgG1 antibody inhibited the ability of human TSLP (hTSLP) to promote TARC release from human PBMCs, and showed better inhibition of human TSLP-promoted TARC release compared to reference antibody AMG157 (described in U.S. Patent No. 7,982,016).

[0063] Table 2. Activity test results of anti-TSLP antibodies

[0064] 2. Preparation and activity detection of anti-FcRn antibodies

[0065] (1) Preparation of anti-FcRn antibodies

[0066] Balb / c mice were immunized with recombinant human FcRn and Freund's adjuvant to establish a mouse scFv antibody phage display library. Through screening, identification and optional humanization, full-length antibodies FcRn-01-IgG4 and FcRn-02-IgG4 targeting human FcRn were finally constructed and prepared. The amino acid sequences of the antibodies are shown in Table 1.

[0067] In addition, RNA was extracted from human blood samples to establish a human natural scFv antibody yeast display library, from which antibodies targeting the human RcRn target were screened. After further optimization, full-length antibodies FcRn-03-IgG4 and FcRn-04-IgG4 were constructed. The amino acid sequences of the antibodies are shown in Table 1.

[0068] (2) Detection of circulating inhibitory activity of anti-FcRn antibodies

[0069] FcRn has the property of pH-dependent binding to IgG antibodies and is a key receptor for maintaining the metabolic level of IgG-type immunoglobulins in the body. MDCK cells are model cells for studying the transport of IgG by FcRn. MDCK cells stably transfected with two subunits of human FcRn (hFCGRT and hB2M) can transport human IgG, and anti-FcRn antibodies can block this activity. The strength of the inhibitory activity of anti-FcRn antibodies is evaluated by measuring the IgG concentration. The results are shown in Table 3. The prepared FcRn antibody can inhibit the circulation of human IgG, and the inhibitory activity on the circulation of IgG is comparable to that of the positive control antibody Rozanolixizumab (UCB, anti-FcRn antibody), or even better than the control antibody.

[0070] Table 3. Antibody activity test results

[0071] Example 2: Screening of dual surfactants in high-concentration antibody preparations

[0072] During the continuous oscillation process, the probability of collision between antibodies is greatly increased. At the same time, the air-solution interface and bottle wall-solution interface will be continuously introduced during the oscillation process, which will make the antibodies at the interface easily form aggregates or even particles.

[0073] This experiment investigated the effect of surfactants on antibody stability under oscillation conditions. The buffer in the prescription was 20 mM acetate, pH 5.2, and the stabilizer was 60 mg / ml sucrose. The surfactant screening prescription design is shown in Table 4. Among them, TSLP antibody takes TSLP-01-IgG1 antibody as an example, and FcRn antibody takes FcRn-01-IgG4 as an example. The stability of different prescriptions was investigated under oscillation conditions (2-8°C, 300 rpm, two months). The test items included concentration, pH, osmotic pressure, biological activity (relative to the labeled amount, %), insoluble particles, and aggregation characteristics. Among them, the insoluble particles were detected by the Flowcam method, and the aggregate characteristics were detected by the SEC-HPLC method and the NR-CE-SDS method. The test results are shown in Tables 5 and 6.

[0074] Table 4. Surfactant screening formulation design scheme

[0075] Table 4. Surfactant screening formulation design scheme (continued)

[0076] Table 5. Results of visible foreign matter and insoluble particles in surfactant formulation screening

[0077] Table 6. Aggregate test results for surfactant formulation screening

[0078] After 2 months of oscillation, the pH of each group of antibody preparations was within the range of 5.21-5.25, the osmotic pressure was within the range of 295-300 mOsm, the concentration was within the range of 150 mg / ml-160 mg / ml, and the biological activity of the protein samples (relative to the labeled amount, %) was within the range of 115-130%, and all indicators were basically stable.

[0079] Antibody preparations with polysorbate or poloxamer added as surfactants showed visible foreign matter at 7 days or 1 month; Flowcam test results showed that at 2 months, compared with 0 months, the number of particles >25μm increased by 113-205, and the number of particles >10μm increased by 302-600; SEC test showed that the main peak of prescriptions 1-3 groups had decreased to below 85% at 10 days, indicating that the prescription preparation produced a large number of aggregates; at 2 months, the main peak of prescriptions 4-10 groups decreased by 4.70-9.20% compared with 0 months; NR-CE test showed that the main peak decreased by 2.5-4.2% at 2 months compared with day 0.

[0080] In contrast, no visible foreign matter was found in the antibody preparations with the addition of two surfactants, polysorbate and poloxamer, after 2 months of oscillation; Flowcam detection showed that at 2 months, compared with 0 months, there were only 14-23 particles >25μm and only 179-201 particles >10μm; SEC-HPLC detection showed that the main peak decreased by 0.18-0.23% at 2 months compared with day 0; NR-CE detection showed that the main peak decreased by 0.3-1.1% at 2 months compared with day 0.

[0081] The above results show that, in high-concentration antibody preparations, under oscillation conditions, a single surfactant (polysorbate or poloxamer) cannot effectively prevent the generation of aggregates, and thus cannot effectively maintain the stability of the antibody. However, after combining two surfactants, polysorbate and poloxamer, in a high-concentration antibody preparation, the stability of the antibody can be maintained more effectively than a single surfactant.

[0082] Example 3: Prescription screening for high concentration antibody preparations

[0083] In this experiment, the formulation of high-concentration antibody preparations was further screened under oscillation conditions. The combination of surfactants in the formulation was 0.3 mg / ml polysorbate 80 and 1.0 mg / ml poloxamer 188 as an example. The screening formulation design is shown in Table 7. The stability of different formulations was investigated under oscillation conditions (2-8°C, 300 rpm, two months). The test items included concentration, pH, osmotic pressure, biological activity (relative to the labeled amount, %), insoluble particles, and aggregation characteristics. Among them, the insoluble particles were detected by the Flowcam method, and the aggregate characteristics were detected by the SEC method and the NR-CE-SDS method. The test results are shown in Tables 8 and 9.

[0084] Table 7. Screening prescription design scheme

[0085] Table 8. Results of visible foreign matter and insoluble particles in prescription screening

[0086] Table 9. Aggregate test results for formulation screening

[0087] After 2 months of oscillation, no foreign matter was visible in the antibody preparations of each group, and the pH, osmotic pressure, protein concentration, and biological activity of the protein samples (relative to the labeled amount) of each group remained basically stable.

[0088] Flowcam test results showed that at 2 months, compared with day 0, the number of particles >25μm in each group of antibody preparations increased by 14-85, and the number of particles >10μm increased by 179-276, both of which were better than the prescription using only a single surfactant. In contrast, the number of particles >25μm in prescriptions 3-5 and 11-13 groups increased by only 14-20, and the number of particles >10μm increased by only 179-193, showing a better stability effect.

[0089] The results of SEC-HPLC showed that at 2 months, the main peak of each group of antibody preparations decreased by 0.18%-4.5% compared with day 0, which was better than the prescription using only a single surfactant. In contrast, the main peak of the antibody preparations in prescriptions 3-5 and 11-13 groups only decreased by 0.18-0.23%, showing a better stability effect. The conclusion shown by the NR-CE test results is also consistent with this.

[0090] In summary, after using the combination of polysorbate and poloxamer surfactants, each group of antibody preparations maintained good stability, especially formulations 3-5 and 11-13, which had better stability effects.

[0091] Example 4: Confirmation of high concentration antibody preparation prescription

[0092] According to the above examples, one of the prescriptions was selected as an example to prepare high-concentration liquid preparations of antibodies with different sequences, and the stability of these antibody preparations under repeated freezing and thawing, oscillation, acceleration, and long-term conditions was tested. Among them, the difference between FcRn-01-IgG4 (S228P) and FcRn-01-IgG4 is that the heavy chain constant region of the former is SEQ ID NO: 13, and the latter is SEQ ID NO: 14.

[0093] Table 10. Confirmation of high concentration antibody prescription

[0094] (1) Repeated freeze-thaw stability test

[0095] The stability of the antibody preparation was tested under freeze-thaw conditions (freeze at -80 °C / thaw at room temperature, 5 cycles). Table 11 shows the test results of each antibody preparation: no visible foreign matter was found within 5 freeze-thaw cycles, and the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparation were basically unchanged compared to 0 freeze-thaw cycles; the main peak of SEC detection changed very little, all within 0.07%; the main peak and change range of NR-CE detection were within 0.5%. The above results show the excellent stability of the formulations of different antibodies of the present invention under freeze-thaw conditions.

[0096] Table 11. Freeze-thaw test results of antibody preparations

[0097] (2) Oscillation stability test:

[0098] The stability of the antibody preparation was tested under oscillating conditions (2-8°C, 300rpm, upright). Table 12 shows the test results of each antibody preparation: each preparation formula also showed excellent stability under oscillating conditions: within 2 months of oscillation, no visible foreign matter was found, and the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparation were basically unchanged relative to day 0; the main peak of SEC detection changed very little, all within 0.30%; the main peak and change range of NR-CE detection were within 0.7%. The above results show the excellent stability of the preparation formula of the antibody of the present invention under oscillating conditions.

[0099] Table 12. Oscillation test results of antibody preparations

[0100] (3) Acceleration stability:

[0101] The stability of the antibody preparations was tested under accelerated conditions (25°C, 2 months). Table 13 shows the test results of each antibody preparation: no visible foreign matter was found within 2 months of acceleration, and the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparations were basically unchanged compared to day 0; the main peak of SEC detection changed slightly, all within 1.09%; the main peak and change range of NR-CE detection were within 1.30%. The above results show that the preparation formula of the antibody of the present invention can maintain excellent stability under accelerated conditions.

[0102] Table 13. Accelerated test results of antibody preparations

[0103] (4) Long-term stability:

[0104] The stability of the antibody preparations was tested under long-term conditions (2-8°C, upright, 6 months). Table 14 shows the test results of each antibody preparation: no visible foreign matter was found under long-term conditions for 6 months, and the concentration, pH, osmotic pressure, biological activity, and insoluble particles of the antibody preparations were basically unchanged compared to day 0; the main peak of SEC detection changed slightly, all within 0.18%; the main peak and change range of NR-CE detection were within 0.4%. The above results show that the preparation prescription of the antibody of the present invention can maintain excellent stability under long-term conditions.

[0105] Table 14. Long-term test results of antibody preparations

[0106] In summary, the high-concentration antibody preparation of the present invention has strong stability under freeze-thaw, oscillation, accelerated and long-term conditions, is suitable for antibodies of various sequences, can maintain good stability during preparation, transportation and storage, and ensures clinical drug safety and quality controllability.

Claims

A high-concentration antibody preparation, characterized in that The antibody preparation comprises a monoclonal antibody and a surfactant, wherein the surfactant is polysorbate and poloxamer, and the concentration of the monoclonal antibody is 100 mg / ml-200 mg / ml, preferably 135 mg / ml-165 mg / ml. The antibody preparation according to claim 1, characterized in that The polysorbate is polysorbate 20 and / or polysorbate 80; preferably, the poloxamer is poloxamer 188. The antibody preparation according to claim 1 or 2, characterized in that The concentration of the polysorbate is 0.02 mg / ml-0.45 mg / ml, preferably 0.08 mg / ml-0.4 mg / ml, and more preferably 0.1 mg / ml-0.3 mg / ml. The antibody preparation according to any one of claims 1 to 3, characterized in that The concentration of the poloxamer is 0.4-1.5 mg / ml, preferably 0.8 mg / ml-1.2 mg / ml, and more preferably 1.0 mg / ml. The antibody preparation according to any one of claims 1 to 4, characterized in that The antibody preparation further comprises a stabilizer. Preferably, the stabilizer is any one or more of sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine, glycine, proline, and lysine; more preferably, the stabilizer is any one or more of sucrose, trehalose, sodium chloride, or arginine. The preparation according to claim 5, characterized in that The stabilizer is: sodium chloride, arginine, glycine, proline or lysine at a concentration of 100mM-180mM, more preferably 130mM-150mM; or sucrose, trehalose, maltose, sorbitol and / or mannitol at a concentration of 40mg / ml-80mg / ml, preferably 50mg / ml-80mg / ml, more preferably 50mg / ml-70mg / ml. The antibody preparation according to any one of claims 1 to 6, characterized in that The antibody preparation further comprises a buffer, preferably, the buffer is a citrate buffer, an acetate buffer and / or a histidine hydrochloride buffer. The antibody preparation according to claim 7, characterized in that The concentration of the buffer is 3mM-50mM; preferably, the concentration of the buffer is 10mM-30mM. The antibody preparation according to any one of claims 1 to 8, characterized in that The pH value of the preparation is 4.8-5.5, preferably 5.0-5.

4. The antibody preparation according to claim 1, characterized in that The antibody concentration is 100 mg / ml-200 mg / ml; the stabilizer is 40 mg / ml-80 mg / ml sucrose and / or trehalose, or the stabilizer is 100 mM-180 mM sodium chloride or arginine; the buffer is 3 mM-50 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 4.8-5.5; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer. The antibody preparation according to claim 1, characterized in that The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-80 mg / ml sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 4.8-5.5; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer. The antibody preparation according to claim 1, characterized in that The antibody concentration is 135 mg / ml-165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45 mg / ml polysorbate and 0.4 mg / ml-1.5 mg / ml poloxamer. The antibody preparation according to claim 1, characterized in that The preparation is any one of the following antibody preparations: (1) the antibody concentration is 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml or 165 mg / ml; the stabilizer is 50 mg / ml-70 mg / ml of sucrose and / or trehalose, or the stabilizer is 130 mM-150 mM sodium chloride or arginine; the buffer is 10 mM-30 mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02 mg / ml-0.45mg / ml polysorbate and 0.4mg / ml-1.5mg / ml poloxamer; (2) the antibody concentration is 135mg / ml-165mg / ml; the stabilizer is 50mg / ml, 60mg / ml or 70mg / ml sucrose and / or trehalose, or the stabilizer is 130mM, 140mM or 150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg / ml- 0.45mg / ml polysorbate and 0.4mg / ml-1.5mg / ml poloxamer; (3) the antibody concentration is 135mg / ml-165mg / ml; the stabilizer is 50mg / ml-70mg / ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine; the buffer is 10mM, 20mM or 30mM acetate buffer, citric acid buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg / ml-0.45mg / ml polysorbate and 0 .4mg / ml-1.5mg / ml poloxamer; (4) the antibody concentration is 135mg / ml-165mg / ml; the stabilizer is 50mg / ml-70mg / ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0, 5.1, 5.2, 5.3 or 5.4; the surfactant is 0.02mg / ml-0.45mg / ml polysorbate and 0.4mg / ml-1.5mg / ml of poloxamer; (5) the antibody concentration is 135mg / ml-165mg / ml; the stabilizer is 50mg / ml-70mg / ml of sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citrate buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg / ml, 0.03mg / ml, 0.04mg / ml, 0.05mg / ml, 0.06mg / ml, 0.08mg / ml, 0.10mg / ml, 0.12mg / ml, 0.15mg / ml, 0.20mg / ml, 0.30mg / ml, 0.40mg / ml or 0.45mg / ml Polysorbate and 0.4mg / ml-1.5mg / ml poloxamer; (6) the antibody concentration is 135mg / ml-165mg / ml; the stabilizer is 50mg / ml-70mg / ml sucrose or trehalose, or the stabilizer is 130mM-150mM sodium chloride or arginine; the buffer is 10mM-30mM acetate buffer, citric acid buffer and / or histidine hydrochloride buffer; the pH value of the preparation is 5.0-5.4; the surfactant is 0.02mg / ml-0.45mg / ml polysorbate and 0.4mg / ml, 0.5mg / ml, 0.6mg / ml, 0.8mg / ml, 1.0mg / ml, 1.2mg / ml or 1.5mg / ml poloxamer; the polysorbate is polysorbate 80 or polysorbate 20. The antibody preparation according to claim 1, characterized in that The antibody preparation is any one of the following preparations: (1) the antibody concentration is 150 mg / ml; the stabilizer is 130 mM sodium chloride; the buffer is 20 mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (2) the antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (3) the antibody concentration is 135mg / ml; the stabilizer is 50mg / ml sucrose; the buffer is 10mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1mg / ml or 0.3mg / ml polysorbate and 1.0mg / ml poloxamer; (4) the antibody concentration is 150mg / ml; the stabilizer is 60mg / ml sucrose; the buffer is 20mM acetate buffer; the pH value of the preparation is 5.2; the surfactant is 0.1mg / ml or 0.3mg / ml polysorbate and 1.0mg / ml poloxamer; (5) the antibody concentration is 165mg / ml; the stabilizer is 70mg / ml sucrose; The buffer is 30 mM acetate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (6) the antibody concentration is 150 mg / ml; the stabilizer is 80 mg / ml sucrose; the buffer is 30 mM acetate buffer; the pH value of the preparation is 5.4; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (7) the antibody concentration is 150 mg / ml; the stabilizer is 150 mM arginine; the buffer is 20 mM acetate buffer; the pH value of the preparation is The concentration of the antibody is 5.2; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (8) the antibody concentration is 160 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 5.0; the surfactant is 0.1 mg / ml or 0.3 mg / ml polysorbate and 1.0 mg / ml poloxamer; (9) the antibody concentration is 150 mg / ml; the stabilizer is 60 mg / ml sucrose; the buffer is 20 mM citrate buffer; the pH value of the preparation is 4.8; the surfactant is 0.1 mg / ml or 0.3mg / ml polysorbate and 1.0mg / ml poloxamer; (10) the antibody concentration is 150mg / ml; the stabilizer is 60mg / ml sucrose; the buffer is 20mM histidine hydrochloride buffer; the pH value of the preparation is 5.5; the surfactant is 0.1mg / ml or 0.3mg / ml polysorbate and 1.0mg / ml poloxamer; (11) the antibody concentration is 150mg / ml; the stabilizer is 60mg / ml sucrose; the buffer is 20mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.02mg / ml or 0.05mg / ml polysorbate and 1. 5mg / ml poloxamer; (12) the antibody concentration is 150mg / ml; the stabilizer is 60mg / ml sucrose; the buffer is 20mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.15mg / ml polysorbate and 0.5mg / ml poloxamer; (13) the antibody concentration is 150mg / ml; the stabilizer is 60mg / ml sucrose; the buffer is 20mM histidine hydrochloride buffer; the pH value of the preparation is 5.2; the surfactant is 0.45mg / ml polysorbate and 0.4mg / ml poloxamer; the polysorbate is polysorbate 80 or polysorbate 20. . The antibody preparation according to any one of claims 1 to 14, characterized in that The antibody formulation further comprises an antioxidant and / or a preservative. The antibody preparation according to any one of claims 1 to 15, characterized in that The antibody preparation is a liquid preparation or a powder for injection. The antibody preparation according to any one of claims 1 to 16, characterized in that The antibody is an anti-TSLP antibody or an anti-FcRn antibody. Use of the antibody preparation according to any one of claims 1 to 17 in the preparation of a medicament for treating a disease; preferably, the disease includes an inflammatory or autoimmune disease.