Anti-TNF-alpha antibody preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of pharmacy, and provides an anti-TNF-alpha antibody preparation and a preparation method and application thereof.The preparation comprises an anti-TNF-alpha antibody and a pharmaceutically acceptable carrier, the anti-TNF-alpha antibody comprises a heavy chain with the amino acid sequence shown as SEQ ID NO.1 and a light chain with the amino acid sequence shown as SEQ ID NO.2, and the heavy chain is a heavy chain with the amino acid sequence shown as SEQ ID NO.1; the pharmaceutically acceptable carrier comprises a stabilizer, a surfactant and a buffer agent. The antibody formulations of the present invention have high stability and do not support microbial growth.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceuticals, and in particular relates to an antibody preparation against tumor necrosis factor alpha (TNF-alpha), a preparation method and use thereof. Background Art

[0002] Tumor necrosis factor α (TNF-α), mainly secreted by macrophages and monocytes, is an important pro-inflammatory factor and immunomodulatory factor that regulates a variety of physiological processes, such as inducing the expression of adhesion molecules, regulating lymphocytes to form lymphoid tissue, and regulating the body's immune defense. Studies have found that when TNF-α is at a low concentration, it is considered beneficial to humans, such as enhancing immune response; when TNF-α is at a high concentration, it can cause inflammatory response and organ damage. For example, a large amount of TNF-α is produced in the body of a sepsis patient in a short period of time, which can cause shock in the patient. Studies have found that TNF-α binds to tumor necrosis factor receptors (TNFR) on the surface of downstream effector cells, induces the expression of adhesion molecules, such as intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), causes neutrophils to aggregate, regulates the proliferation, maturation and activation of neutrophils and macrophages, and further stimulates monocytes and vascular endothelial cells to release other cytokines, such as proinflammatory factors interleukin factor 1 (IL-1) and interleukin factor 8 (IL-8), triggering a cascade reaction and ultimately leading to inflammatory damage to tissues (Paul AT, Gohil VM, Bhutani K K. Modulating TNF-alpha signaling with natural products. Drug Discovery Today, 2006, 11 (11): 725-32.). Elevated TNF-α concentrations were found in the blood and synovium of patients with multiple chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis and ankylosing spondylitis, indicating that TNF-α is closely related to these diseases. In recent years, many newly developed biological agents, such as TNF-α inhibitors, can control the progression of the disease by blocking or downregulating the activity of TNF-α. The main mechanism of action of TNF-α inhibitors is: after specifically binding to secreted tumor necrosis factor α (sTNF-α), it can block its binding with TNFR and inhibit its biological activity; it can also bind to transmembrane tumor necrosis factor α (tmTNF-α) on TNF-α synthesizing cells, activate the reverse signaling pathway to reduce the amount of TNF-α synthesis, and even cause apoptosis of TNF-α synthesizing cells, thereby alleviating the patient's inflammatory symptoms.

[0003] At present, the TNF-α inhibitor drugs on the market mainly include Infliximab (trade name ), Etanercept (trade name ), Adalimumab (trade name ), Golimumab (trade name Simponi ) and Certolizumab pegol (trade name ). Among them, golimumab is a fully human monoclonal antibody, and the subcutaneous injection preparation approved for marketing is the first TNF-α inhibitor drug approved internationally for subcutaneous administration once every 4 weeks.

[0004] From the initial mouse-derived antibodies to the currently widely used fully human antibodies, the immunogenicity of antibodies has been greatly reduced. However, mammalian cells can synthesize Neu5Gc sialic acid (NGNA) that is immunogenic to humans. For example, when Sp2 / 0 cells are used for antibody production, the level of antibody glycosylation modification by NGNA is high, which may pose a risk of causing an immune response in the human body.

[0005] Therefore, there is still a need in the art for anti-TNF-α antibodies and formulations thereof with improved properties. Summary of the invention

[0006] In one aspect, the present invention provides an anti-TNF-α antibody product, in some embodiments, the anti-TNF-α antibody product is an anti-TNF-α antibody preparation. The preparation has improved performance, excellent antibody protein stability and preparation stability at high concentrations of antibody protein (especially maintaining excellent antibody protein stability under conditions such as high temperature, light changes, repeated freezing and thawing), thereby reducing the risk of adverse reactions, and is not prone to the growth of microorganisms. The preparation comprises an anti-TNF-α antibody and a pharmaceutically acceptable carrier.

[0007] In some embodiments, the anti-TNF-α antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO.1 and a light chain having an amino acid sequence as shown in SEQ ID NO.2.

[0008] In some embodiments, the pharmaceutically acceptable carrier comprises a stabilizer; in some embodiments, the stabilizer is a sugar, arginine hydrochloride, glycine, methionine, or a combination thereof; in some embodiments, the stabilizer is trehalose or sucrose; in some embodiments, the stabilizer is trehalose. In some embodiments, the antibody formulation does not comprise a sugar alcohol.

[0009] In some embodiments, the concentration of the anti-TNF-α antibody is 5-130 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is 50-130 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is 90-110 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is 100 mg / ml.

[0010] In some embodiments, the concentration of the stabilizer is 160-265 mM; in some embodiments, the concentration of the stabilizer is 210-240 mM; in some embodiments, the concentration of the stabilizer is 225 mM. In some embodiments, the stabilizer is 225 mM trehalose.

[0011] In some embodiments, the antibody preparation may be in liquid form, powder form or any other suitable form. Depending on the administration route, storage conditions and other requirements, the powder form may be resuspended into a solution form before use.

[0012] In some embodiments, the pharmaceutically acceptable carrier further comprises a surfactant and / or a buffer.

[0013] In some embodiments, the surfactant is polysorbate; in some embodiments, the surfactant is polysorbate 20 and / or polysorbate 80; in some embodiments, the surfactant is polysorbate 80; in some embodiments, the concentration of the surfactant is 0.05-0.5 mg / ml; in some embodiments, the concentration of the surfactant is 0.1-0.5 mg / ml; in some embodiments, the concentration of the surfactant is 0.1-0.3 mg / ml; in some embodiments, the concentration of the surfactant is 0.2 mg / ml. In some embodiments, the surfactant is 0.2 mg / ml of polysorbate 80.

[0014] In some embodiments, the pH of the antibody formulation is 4.5-6.5; in some embodiments, the pH is 4.5-6.0; in some embodiments, the pH is 5.0-6.0; in some embodiments, the pH is 5.0-5.8; in some embodiments, the pH is 5.2-5.8; in some embodiments, the pH is 5.5.

[0015] In some embodiments, the antibody formulation comprises a solvent. In some embodiments, the solvent is, for example, water, such as water for injection.

[0016] In some embodiments, the buffer of the antibody formulation is one or more of a histidine buffer, a glutamate buffer, a sodium acetate buffer, a succinate buffer, and a citrate buffer; in some embodiments, the buffer is a histidine buffer, a glutamate buffer, a sodium acetate buffer; in some embodiments, the buffer is a histidine buffer. In some embodiments, the concentration of the buffer is 5-30 mM; in some embodiments, the concentration of the buffer is 10-20 mM; in some embodiments, the concentration of the buffer is 10 mM. In some embodiments, the buffer is a 10 mM histidine buffer.

[0017] Various pharmaceutically acceptable carriers of the antibody preparation may be provided together with the antibody, or may be provided separately and mixed before use.

[0018] In some embodiments, an anti-TNF-α antibody preparation is provided, the antibody preparation comprising solvent water, an anti-TNF-α antibody, a surfactant, a stabilizer and a buffer; the anti-TNF-α antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO.1 and a light chain having an amino acid sequence as shown in SEQ ID NO.2, and the antibody preparation comprises 5-130 mg / ml of the anti-TNF-α antibody, 160-265 mM of the stabilizer, 0.05-0.5 mg / ml of the surfactant, 5-30 mM of the buffer, and the pH of the antibody preparation is 4.5-6.5. In some embodiments, the antibody preparation comprises 90-110 mg / ml of the anti-TNF-α antibody, 210-240 mM of the stabilizer, 0.1-0.3 mg / ml of the surfactant, 10-20 mM of the buffer, and the pH of the antibody preparation is 4.5-6.0. In some embodiments, the antibody preparation comprises 90-110 mg / ml of the anti-TNF-α antibody, 210-240 mM trehalose, 0.1-0.3 mg / ml polysorbate 80, 10-20 mM histidine buffer, and the pH of the antibody preparation is 4.5-6.0. In some embodiments, the antibody preparation comprises 100 mg / ml of the anti-TNF-α antibody, 225 mM trehalose, 0.2 mg / ml polysorbate 80, 10 mM histidine buffer, and the pH of the antibody preparation is 5.0-5.8.

[0019] In some embodiments, an anti-TNF-α antibody preparation is provided, which comprises solvent water, an anti-TNF-α antibody, a surfactant, a stabilizer and a buffer; the anti-TNF-α antibody comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO.1 and a light chain with an amino acid sequence as shown in SEQ ID NO.2, and the concentration of the anti-TNF-α antibody is 100 mg / ml, the stabilizer is 85 mg / ml trehalose dihydrate, the surfactant is 0.2 mg / ml polysorbate 80, the antibody preparation has a pH of 5.5, and the pH is maintained by adding 1.72 mg / ml histidine hydrochloride and 0.28 mg / ml histidine to the liquid component of the antibody preparation.

[0020] In a second aspect, the present invention provides a method for preparing the anti-TNF-α antibody preparation as described above.

[0021] In one embodiment, a method for preparing a liquid anti-TNF-α antibody formulation as described above is provided, the method comprising:

[0022] Weigh stabilizers, surfactants and buffers,

[0023] The weighed components are dispersed in a liquid solvent for injection to prepare a solvent system.

[0024] The solvent system is mixed with the anti-TNF-α antibody under stirring.

[0025] In some embodiments, in the method, the buffer can be one or more of a histidine buffer, a glutamate buffer, a sodium acetate buffer, a succinate buffer, and a citrate buffer; the stabilizer can be a sugar, arginine hydrochloride, glycine, methionine, or a combination thereof; and the surfactant can be a polysorbate.

[0026] In some embodiments, a method for preparing an anti-TNF-α antibody formulation is provided, the method comprising:

[0027] Weigh trehalose, polysorbate 80, histidine, and histidine hydrochloride;

[0028] Dissolving the weighed components in water for injection, thereby preparing a solvent system;

[0029] mixing the solvent system with the anti-TNF-α antibody under stirring;

[0030] The final concentrations of the components are 90-110 mg / ml anti-TNF-α antibody, 80-90 mg / ml trehalose, 0.1-0.3 mg / ml polysorbate 80, 1-3 mg / ml histidine hydrochloride and 0.2-0.5 mg / ml histidine, respectively, and the final pH of the antibody preparation is 5.0-6.0.

[0031] In some embodiments, the final concentrations of the components are 100 mg / ml anti-TNF-α antibody, 85 mg / ml trehalose, 0.2 mg / ml polysorbate 80, 1.72 mg / ml histidine hydrochloride, and 0.28 mg / ml histidine.

[0032] The method may further include the steps of preparing an anti-TNF-α antibody preparation and / or sterilizing and packaging the antibody preparation, for example, sterilizing the prepared antibody preparation by passing it through a 0.22 μm pore size hydrophilic polyvinylidene fluoride or polyethersulfone filter membrane.

[0033] In a third aspect, the present invention provides a method for treating TNF-α-related diseases, comprising administering the anti-TNF-α antibody preparation of the present invention as described above to a patient in need thereof. The TNF-α-related diseases include, but are not limited to, chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, and ankylosing spondylitis.

[0034] In some embodiments, the method comprises administering the antibody formulation of the invention by subcutaneous injection, in some embodiments, once every 4 weeks; in some embodiments, 50 mg / time per administration.

[0035] In a fourth aspect, the present invention provides an antibody pharmaceutical product for treating TNF-α-related diseases, comprising the anti-TNF-α antibody preparation of the present invention as described above and a container for storing the preparation. The pharmaceutical product may also include instructions for use. The container may be any container conventionally used in the art for storing drugs, such as a prefilled container, a prefilled syringe, a vial, an ampoule, a pouch, etc.

[0036] In a fifth aspect, the present invention provides the use of the anti-TNF-α antibody preparation or antibody pharmaceutical product of the present invention as described above in the preparation of a medicament for treating TNF-α-related diseases. The TNF-α-related diseases include, but are not limited to, chronic inflammatory diseases, such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, and ulcerative colitis.

[0037] In a sixth aspect, the present invention provides an anti-TNF-α antibody containing a low level of Neu5Gc sialic acid (NGNA), which reduces the risk of adverse reactions while ensuring that the activity of the antibody is not affected. The anti-TNF-α antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises an amino acid sequence as shown in SEQ ID NO.1, and the light chain comprises an amino acid sequence as shown in SEQ ID NO.2. In some embodiments, the molar ratio of the Neu5Gc sialic acid (NGNA) level of the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.1. In some embodiments, the anti-TNF-α antibody is expressed by a CHO cell containing a nucleic acid or vector encoding the anti-TNF-α antibody. In some embodiments, the CHO cell is a CHO-K1 cell. In some embodiments, the CHO-K1 cell is domesticated to a CHO-K1 cell adapted to suspension culture.

[0038] In a seventh aspect, the present invention provides a method for preparing an anti-TNF-α antibody of the present invention. In some embodiments, the method uses a CHO cell line as a host cell. In some embodiments, the method comprises culturing a CHO cell containing a nucleic acid or vector encoding the anti-TNF-α antibody, so that the CHO cell expresses the anti-TNF-α antibody. In some embodiments, the method comprises the following steps: 1) transfecting a nucleic acid or vector encoding the anti-TNF-α antibody into a CHO cell; 2) culturing the CHO cell so that the CHO cell expresses the anti-TNF-α antibody; in some embodiments, the anti-TNF-α antibody comprises a heavy chain and a light chain, and its heavy chain amino acid sequence is shown in SEQ ID NO: 1, and the light chain amino acid sequence is shown in SEQ ID NO: 2. In some embodiments, the CHO cell is a CHO-K1 cell.

[0039] In some embodiments, the method further comprises the step of: harvesting the culture fluid after stopping the culture. In some embodiments, the method further comprises purifying the anti-TNF-α antibody after harvesting the culture fluid.

[0040] In the seventh aspect, the present invention provides an anti-TNF-α antibody prepared by the above method, wherein the antibody has a reduced deamidation level and / or a reduced NGNA glycosylation level. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) in the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.1; in some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.01; in some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.005.

[0041] In some embodiments, the anti-TNF-α antibody expressed by the CHO cells comprises a heavy chain amino acid sequence as shown in SEQ ID NO.1, a light chain amino acid sequence as shown in SEQ ID NO.2, and one or more of the relative affinity for binding to TNF-α, the ability to bind to TNF-α, the inhibitory effect on the biological activity of TNF-α, the relative affinity for binding to FcRn, the relative affinity for binding to FcγRIIIa (F158), the relative affinity for binding to FcγRIIIa (V158), and the pharmacokinetic characteristics in cynomolgus monkeys (such as plasma drug concentration) are substantially consistent with those of simponi (the difference does not exceed ±20%, or ±10%, or ±5%, or does not exceed 3 times, 2 times, or 1 times the standard deviation). In some embodiments, the anti-TNF-α antibody expressed by the CHO cells is antibody A.

[0042] In an eighth aspect, the present invention provides an antibody preparation comprising the anti-TNF-α antibody as described above. In some embodiments, the antibody preparation further comprises a pharmaceutically acceptable carrier.

[0043] The anti-TNF-α antibody developed by the present invention has a low NGNA glycosylation level under the condition of ensuring the activity of the antibody, so that the antibody has high activity and low immunogenicity. The anti-TNF-α antibody preparation developed by the present invention contains a high concentration of the anti-TNF-α antibody protein of the present invention, the antibody has high activity and low immunogenicity, and the high concentration antibody protein contained therein is stable, especially resistant to protein aggregation caused by high temperature, light, and repeated freezing and thawing, and does not support microbial growth. Therefore, the anti-TNF-α antibody preparation of the present invention meets the needs of the art for anti-TNF-α antibody pharmaceutical preparations with improved performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The graph shows the size exclusion chromatography test results of the buffer screening test of the anti-TNF-α antibody preparation tested in Example 3.

[0045] Figure 2 The capillary electrophoresis detection result of the stability study of the anti-TNF-α antibody preparation of the present invention under high temperature (40° C.) conditions is shown.

[0046] Figure 3 The graph shows the size exclusion chromatography test results of the freeze-thaw stability study of the anti-TNF-α antibody preparation of the present invention.

[0047] Figure 4 The relative affinities of the Antibody A preparation of the present invention and Simponi for binding to TNF-α are shown.

[0048] Figure 5The relative binding abilities of the antibody A preparation of the present invention and Simponi to TNF-α are shown.

[0049] Figure 6 The results show that the antibody A preparation of the present invention and Simponi inhibit the biological activity of TNF-α.

[0050] Fig. 7A The effect of the antibody A of the present invention on the arthritis score of the Tg197 transgenic spontaneous arthritis mouse model is shown.

[0051] Figure 7B The effect of the antibody A of the present invention on the histopathological score of the Tg197 transgenic spontaneous arthritis mouse model is shown.

[0052] Fig. 8A and Figure 8B The molecular weight comparison of the antibody A of the present invention and the antibody of Simponi is shown ( Fig. 8A : intact molecular weight; Figure 8B : desugared molecular weight).

[0053] Fig. 9 The relative affinities of antibody A of the present invention and the antibody of Simponi for binding to FcRn are shown.

[0054] Fig.10 The relative affinities of the antibody A of the present invention and the antibody of Simponi for binding to FcγRIIIa (F158) are shown.

[0055] Fig.11 The relative affinities of the antibody A of the present invention and the antibody of Simponi for binding to FcγRIIIa (V158) are shown.

[0056] Fig. 12A The pharmacokinetic comparison of the antibody A of the present invention and the low-dose group of the antibody of Simponi at 3 mg / kg in cynomolgus monkeys is shown.

[0057] Fig. 12B The pharmacokinetic comparison of the antibody A of the present invention and the high-dose group of Simponi at 10 mg / kg in cynomolgus monkeys is shown. DETAILED DESCRIPTION

[0058] In order to be able to more clearly understand the technical content of the present invention, the following examples are specifically described in detail. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art can know or only determine various equivalents of the specific embodiments of the present invention through routine experiments. These equivalents are also included in the scope of the present invention. All documents and patents cited in the present invention are incorporated herein by reference. The experimental methods for which specific conditions are not specified in the following examples are usually based on conventional conditions or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0059] It will be appreciated by the skilled person that the weights and / or weight to volume ratios mentioned herein can be converted to moles and / or molar concentrations using the known molecular weights of the corresponding ingredients. The weights cited in the examples are for the corresponding volumes. It will be appreciated by the skilled person that the weights can be adjusted proportionally when different formulation volumes are desired.

[0060] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0061] definition

[0062] As used herein, in the present invention, "%" of a component specifically refers to weight-volume (w / v) percentage, wherein the weight unit may be g and the volume unit may be ml. For example, 1% stabilizer in a solution means that 100 ml of the solution contains 1 g of stabilizer, or the stabilizer content is 0.01 g / ml.

[0063] "About" or "approximately" refers to the conventional error range of the corresponding numerical value that is easily known to those skilled in the relevant technical field. In some embodiments, "about" or "approximately" mentioned herein refers to the described numerical value and its ±10%, ±5%, ±1% or ±0.1% range.

[0064] "Including" or "comprising" means that compositions and methods, etc. include the listed elements (such as components in the composition, steps in the method, etc.), but do not exclude others. When "consisting essentially of..." is used to define compositions and methods, it means excluding other elements that have a fundamental impact on the combination for the intended use, but does not exclude elements that do not substantially affect the characteristics of the composition or method. "Consisting of..." means excluding elements that are not specifically listed. The embodiments defined by each of these transition terms are within the scope of the present invention. For example, when a composition is described as including components A, B and C, a composition consisting essentially of A, B and C and a composition consisting of A, B and C are independently within the scope of the present invention.

[0065] As used herein, "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule having a biological activity that binds to an antigen. Such examples include, but are not limited to, a heavy chain or light chain or its ligand-binding portion's complementary determining region (CDR), a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a conjugated protein.

[0066] As used herein, the term "treatment" refers to both therapeutic treatment and preventive measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or conditions, such as the progression of TNF-α related diseases. Beneficial or desired clinical results include, but are not limited to, relieving symptoms, reducing the extent of the disease, stabilizing the condition (i.e., not worsening), delaying or slowing disease progression, improving or alleviating the disease state, alleviating (partially or completely), whether detectable or undetectable. People in need of treatment include those patients who already have a condition or condition, as well as those who are prone to a condition or condition, or those patients who need to prevent a condition or condition.

[0067] As used herein, "patient" refers to any subject, particularly a mammalian subject, for whom treatment is desired. Mammalian subjects include humans, domestic animals, livestock, zoo animals, sports animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0068] As used herein, "a patient in need thereof" includes patients who would benefit from administration of an antibody or formulation of the invention, such as a mammalian patient.

[0069] As used herein, the term "pharmaceutically acceptable" refers to drugs listed in recognized pharmacopoeias for use in animals, especially in humans."Pharmaceutically acceptable carriers" are generally non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or any type of formulation aids.

[0070] The term "buffer", in some literature, is also referred to as a buffer system or buffer system, which includes but is not limited to organic acid salts, such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or phthalic acid and their salts; Tris, thomethamine hydrochloride, or phosphate buffer. In addition, amino acids and their salts can also be used as buffers. Such amino acid components include but are not limited to glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid or aspartic acid. In some embodiments, the buffer is a histidine salt buffer.

[0071] The amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system that constitutes the buffer. In some embodiments, molar concentration is used as the unit of the amount of buffer (or buffer), and its value refers to the molar concentration of the buffer pair in the buffer system of the buffer (or buffer). For example, when a histidine buffer composed of histidine and histidine hydrochloride is used as a buffer, a given concentration of histidine salt buffer (such as 10mM) is the combined concentration of histidine and histidine hydrochloride (such as 5mM histidine and 5mM histidine hydrochloride; or 1.8mM histidine and 8.2mM histidine hydrochloride).

[0072] The term "stabilizer" includes, but is not limited to, monosaccharides such as fructose, maltose, galactose, glucose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; ionic stabilizers, which include salts such as NaCl or amino acid components such as arginine-HCl, proline, glycine, methionine.

[0073] The term "surfactant" includes, but is not limited to, polysorbates (such as polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine, or cetyl betaine; lauryl Amidopropyl betaine, cocoamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitopropyl betaine or isostearamidopropyl betaine (e.g. lauroamidopropyl); myristamidopropyl dimethylamine, palmitopropyl dimethylamine or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68, etc.), etc. In some embodiments, the surfactant is polysorbate 80.

[0074] The term "pharmaceutically acceptable" refers to materials for animals, especially humans, that are approved by a national regulatory agency or listed in a nationally promulgated pharmacopoeia or other generally recognized pharmacopoeia. In addition, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation adjuvant, etc.

[0075] Anti-TNF-α Antibodies

[0076] The present invention provides an anti-TNF-α antibody with high affinity for TNF-α protein. The anti-TNF-α antibody disclosed in the present invention comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is shown in SEQ ID NO:1, or a sequence having 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence SEQ ID NO:1; and the light chain amino acid sequence is shown in SEQ ID NO:2, or a sequence having 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence SEQ ID NO:2.

[0077] The sequence of SEQ ID NO:1 is:

[0078] QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

[0079] The sequence of SEQ ID NO:2 is as follows:

[0080] EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0081] In some embodiments, the anti-TNF-α antibody is golimumab( or a biosimilar thereof (such as BOW100, ONS-3035, or Antibody A)).

[0082] In some embodiments, the anti-TNF-α antibody is prepared by the following method: culturing CHO cells transfected with nucleic acid or vector encoding anti-TNF-α antibody, so that the CHO cells express the anti-TNF-α antibody. In some embodiments, the CHO cells are CHO-K1 cells. In some embodiments, the CHO-K1 cells are CHO-K1 cells adapted to suspension culture. In some embodiments, the vector can be a plasmid vector, a phage vector, a viral vector, a non-viral vector or a minicircular DNA. In some embodiments, culturing CHO cells includes: under suitable conditions and culture medium, causing the CHO cells to express the anti-TNF-α antibody.

[0083] In some embodiments, the step of culturing CHO cells comprises: culturing for a period of time under suitable conditions and culture medium, so that the CHO cells express the heavy chain and light chain of the antibody and form anti-TNF-α antibodies. In some embodiments, the method further comprises the following steps: stopping the culture and harvesting the culture fluid. In some embodiments, the method further comprises purifying the anti-TNF-α antibody after harvesting the culture fluid.

[0084] In some embodiments, after the culture fluid is harvested, the anti-TNF-α antibody is isolated by centrifuging the culture fluid. In some embodiments, the isolated anti-TNF-α antibody is purified by conventional means, such as by affinity chromatography, anion exchange chromatography and / or cation exchange chromatography; in some embodiments, the purity of the purified anti-TNF-α antibody can reach about 90% or more, about 92% or more, about 94% or more, about 96% or more, 98% or more, or about 99% or more.

[0085] Glycosylation is one of the key quality attributes of antibodies. The realization of the function of monoclonal antibody drugs is closely related to their glycosylation. Glycosylation modification will affect the performance of proteins, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity. Glycosylation can be divided into N-glycosylation and O-glycosylation according to the modification site of glycosylation. N-glycosylation is the most common glycosylation in immunoglobulins secreted by animal cells, and antibody glycosylation is also mainly N-glycosylation. For golimumab, glycosylation mostly occurs at Asn-306. According to the different fine structures of the glycosylation ends (length, branching and monosaccharide arrangement), it can be divided into complex, hybrid and high mannose types, which are closely related to cell species and cell culture conditions.

[0086] From the initial mouse-derived antibodies to the currently widely used fully human antibodies, the immunogenicity of antibodies has been greatly reduced. However, mammalian cells can synthesize Neu5Gc sialic acid (NGNA) that is immunogenic to humans. For example, when Sp2 / 0 cells are used for antibody production, the level of glycosylated NGNA is high, which may pose a risk of causing an immune response in the human body.

[0087] The present invention provides an anti-TNF-α antibody with low NGNA level, the anti-TNF-α antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO.1, and the light chain comprising the amino acid sequence shown in SEQ ID NO.2. In some embodiments, the molar ratio of the Neu5Gc sialic acid (NGNA) level of the anti-TNF-α antibody to the anti-TNF-α antibody is no more than 0.1. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) in the anti-TNF-α antibody to the anti-TNF-α antibody is no more than about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03 or about 0.02. In some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody is no more than about 0.01, no more than about 0.009, no more than about 0.008, no more than about 0.007, no more than about 0.006, no more than about 0.005, or no more than about 0.0045. In some embodiments, the anti-TNF-α antibody is expressed by CHO cells containing a nucleic acid or vector encoding the anti-TNF-α antibody.

[0088] The present invention provides a method for producing the anti-TNF-α antibody of the present invention, comprising the following steps: preparing a nucleic acid or vector encoding the anti-TNF-α antibody; transfecting the nucleic acid or vector encoding the anti-TNF-α antibody into CHO-K1 cells; allowing the CHO-K1 cells to express the anti-TNF-α antibody under appropriate conditions and culture medium; stopping the culture; harvesting the culture fluid; separating the anti-TNF-α antibody by centrifuging the culture fluid; preliminarily purifying the anti-TNF-α antibody obtained by centrifugation by affinity chromatography; further purifying the preliminarily purified anti-TNF-α antibody by anion and cation exchange chromatography to obtain the purified anti-TNF-α antibody. In some embodiments, the present invention provides antibody A; antibody A is an anti-TNF-α antibody, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain is shown in SEQ ID NO: 2; and antibody A is produced by the above method.

[0089] The present invention provides an anti-TNF-α antibody prepared by the above method, wherein the antibody has a lower NGNA glycosylation level. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) in the anti-TNF-α antibody to the anti-TNF-α antibody is no more than about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03 or about 0.02. In some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody is no more than about 0.01, no more than about 0.009, no more than about 0.008, no more than about 0.007, no more than about 0.006, no more than about 0.005 or no more than about 0.0045.

[0090] Dosage form

[0091] The antibody preparations of the present invention can be liquid solutions (e.g., injectable, infusible, perfusible solutions), or powders. The form depends on the desired mode of administration and therapeutic application. The preparation is in the form of an injectable, infusible or perfusible solution. The mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular administration). In some embodiments, the antibody is administered by intravenous infusion or injection. In other embodiments, the antibody is administered by intramuscular or subcutaneous injection.

[0092] Supplementary active compounds can also be incorporated into the pharmaceutical preparation. In some embodiments, the antibody of the present invention or its antigen-binding portion thereof is co-formulated and / or co-administered with one or more other therapeutic drugs. For example, other therapeutic drugs can be DMARDs, NSAIDs, other antibodies that can bind other targets (for example, antibodies that can bind other cytokines or can bind cell surface molecules), cytokines, soluble TNF-α receptors, chemicals that can inhibit TNF-α production and activity, or any combination thereof. In addition, the antibody of the present invention can be used in combination with the above-mentioned therapeutic substances. This combination therapy can advantageously use a lower dose of the administered therapeutic drug, thereby avoiding possible side effects, complications, or low-level patient reactions associated with various monotherapies, or increasing the efficacy.

[0093] In some embodiments, the antibody preparation is an anti-TNF-α antibody preparation. In some embodiments, the anti-TNF-α antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO.1 and a light chain having an amino acid sequence as shown in SEQ ID NO.2. In some embodiments, the concentration of the anti-TNF-α antibody is about 5-130 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is about 50-130 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is about 90-110 mg / ml; in some embodiments, the concentration of the anti-TNF-α antibody is about 100 mg / ml. In some embodiments, the concentration of the anti-TNF-α antibody is about 5 mg / ml, about 10 mg / ml, about 30 mg / ml, about 50 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, or a range between any two concentrations, including endpoint values.

[0094] In some embodiments, the anti-TNF-α antibody in the anti-TNF-α antibody preparation is an anti-TNF-α antibody expressed by CHO cells. In some embodiments, the CHO cells expressing the anti-TNF-α antibody are CHO-K1 cells.

[0095] In some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody in the preparation does not exceed 0.1; in some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.01; in some embodiments, the molar ratio of NGNA in the anti-TNF-α antibody to the anti-TNF-α antibody does not exceed 0.005.

[0096] In some embodiments, the pharmaceutically acceptable carrier comprises a stabilizer; in some embodiments, the stabilizer is a sugar, arginine hydrochloride, glycine, methionine, or a combination thereof; in some embodiments, the stabilizer is trehalose or sucrose; in some embodiments, the stabilizer is trehalose. In some embodiments, the concentration of the stabilizer is about 160-265 mM; in some embodiments, the concentration of the stabilizer is about 210-240 mM; in some embodiments, the concentration of the stabilizer is about 225 mM. In some embodiments, the stabilizer is about 225 mM trehalose. In some embodiments, the anti-stabilizer is trehalose or sucrose, and the concentration of the stabilizer is about 160 mM, about 180 mM, about 200 mM, about 210 mM, about 225 mM, about 240 mM, about 250 mM, about 265 mM, or a range between any two concentrations, including endpoint values.

[0097] In a solid state, trehalose is usually present as trehalose dihydrate, so in some embodiments, trehalose dihydrate is used when preparing a preparation, and a corresponding amount of trehalose in other forms can also be used to prepare, and the resulting preparation contains the same concentration of trehalose. When trehalose dihydrate is used herein to describe the content of trehalose in a preparation, the preparation can contain the said amount of trehalose dihydrate or a corresponding amount of trehalose or other forms of trehalose or a combination thereof, and vice versa.

[0098] In some embodiments, the concentration of the stabilizer is about 60-100 mg / ml; in some embodiments, the concentration of the stabilizer is about 80-90 mg / ml; in some embodiments, the concentration of the stabilizer is about 85 mg / ml. In some embodiments, the stabilizer is about 85 mg / ml of trehalose dihydrate. In some embodiments, the anti-stabilizer is trehalose dihydrate or sucrose, and the concentration of the stabilizer is about 60 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 95 mg / ml, about 100 mg / ml, or a range between any two concentrations, including endpoint values.

[0099] In some embodiments, the pharmaceutically acceptable carrier further comprises a surfactant and / or a buffer.

[0100] In some embodiments, the formulation further comprises a surfactant. In some embodiments, the surfactant comprises a nonionic surfactant, such as polysorbate 80. The surfactant reduces aggregation of the antibody and / or reduces the formation of particles in the formulation and / or reduces adsorption. In some embodiments, the surfactant is polysorbate; in some embodiments, the surfactant is polysorbate 20 and / or polysorbate 80; in some embodiments, the surfactant is polysorbate 80; in some embodiments, the concentration of the surfactant is about 0.05-0.5 mg / ml; in some embodiments, the concentration of the surfactant is about 0.1-0.5 mg / ml; in some embodiments, the concentration of the surfactant is about 0.1-0.3 mg / ml; in some embodiments, the concentration of the surfactant is about 0.2 mg / ml. In some embodiments, the surfactant is about 0.2 mg / ml of polysorbate 80. In some embodiments, the concentration of polysorbate 80 is about 0.05 mg / ml, about 0.1 mg / ml, about 0.15 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, or a range between any two concentrations, including the endpoints.

[0101] In some embodiments, the pH of the antibody formulation is about 4.5-6.5; in some embodiments, the pH is about 4.5-6.0; in some embodiments, the pH is about 5.0-6.0; in some embodiments, the pH is about 5.2-5.8; in some embodiments, the pH is about 5.5. In some embodiments, the pH is about 4.5, about 5.0, about 5.2, about 5.5, about 5.8, about 6.0, or any range between two pH values, including the endpoints.

[0102] In some embodiments, the buffer of the antibody formulation is a histidine buffer, a glutamate buffer, a sodium acetate buffer, a succinate buffer, or a citrate buffer; in some embodiments, the buffer is a histidine buffer, a glutamate buffer, a sodium acetate buffer; in some embodiments, the buffer is a histidine buffer. In some embodiments, the concentration of the buffer is about 5-30 mM; in some embodiments, the concentration of the buffer is about 10-20 mM; in some embodiments, the concentration of the buffer is about 10 mM. In some embodiments, the buffer is a histidine buffer of about 10 mM. In some embodiments, the concentration of the histidine buffer is about 5 mM, about 10 mM, about 20 mM, about 30 mM, or a range between any two concentrations, including endpoint values.

[0103] In some embodiments, the antibody formulation comprises about 5-130 mg / ml anti-TNF-α antibody; about 160-265 mM stabilizer; about 0.05-0.5 mg / ml surfactant; about 5-30 mM buffer; the pH of the antibody formulation is about 4.5-6.5. In some embodiments, the antibody formulation comprises about 90-110 mg / ml anti-TNF-α antibody; about 210-240 mM stabilizer; about 0.1-0.3 mg / ml surfactant; about 10-20 mM buffer; the pH of the antibody formulation is about 4.5-6.0. In some embodiments, the antibody formulation comprises about 90-110 mg / ml anti-TNF-α antibody; about 210-240 mM trehalose; about 0.1-0.3 mg / ml polysorbate 80; about 10-20 mM histidine buffer; the pH of the antibody formulation is about 4.5-6.0. In some embodiments, the antibody formulation comprises about 100 mg / ml anti-TNF-α antibody; about 225 mM trehalose; about 0.2 mg / ml polysorbate 80; about 10 mM histidine buffer; the pH of the antibody formulation is 5.0-5.8. In some embodiments, the antibody formulation comprises about 100 mg / ml anti-TNF-α antibody; about 10 mM-about 142 mM arginine hydrochloride; about 0.2 mg / ml polysorbate 80; about 10 mM histidine buffer; the pH of the antibody formulation is 5.0-5.8. In some embodiments, the antibody formulation comprises about 100 mg / ml anti-TNF-α antibody; about 333 mM glycine; about 0.2 mg / ml polysorbate 80; about 10 mM histidine buffer; the pH of the antibody formulation is 5.0-5.8. In some embodiments, the antibody formulation comprises about 100 mg / ml anti-TNF-α antibody; about 34 mM methionine; about 0.2 mg / ml polysorbate 80; about 10 mM histidine buffer; and the pH of the antibody formulation is 5.0-5.8. In some embodiments, the anti-TNF-α antibody is (golimumab) or its biosimilars (such as BOW100, ONS-3035 or Antibody A).

[0104] In some embodiments, the antibody formulation comprises: about 100 mg / ml Antibody A, about 1.72 mg / ml histidine hydrochloride, about 0.28 mg / ml histidine, about 85 mg / ml trehalose dihydrate, about 0.2 mg / ml polysorbate 80, and a pH of about 5.5.

[0105] In some embodiments, the content of acidic antibody variants in the anti-TNF-α antibody preparation is no more than about 40%, no more than about 35%, no more than about 30%. In some embodiments, the content of acidic antibody variants in the anti-TNF-α antibody preparation is about 15%, about 16%, about 17%, about 18%, about 19%, 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29% or about 30%, or a range between any two concentrations, including endpoints.

[0106] In some embodiments, after the anti-TNF-α antibody preparation provided by the present invention is placed at high temperature (40°C ± 3°C) for 1 week, 2 weeks, 3 weeks or 4 weeks, the acid peak content of the anti-TNF-α antibody does not exceed 30%.

[0107] The present invention provides a liquid aqueous pharmaceutical preparation, including an antibody suitable for therapeutic use, mainly used to treat conditions caused by TNF-α. This pharmaceutical preparation is easy to apply, contains a high protein concentration, and can be injected subcutaneously. In some embodiments, the pharmaceutical preparation has the effect of enhancing stability and can keep the deamidation of the antibody at a low level. In other embodiments, the preparation of the present invention is stable after at least 5 freeze-thaw cycles. In other embodiments, the preparation of the present invention remains stable after being placed at high temperature (40° C.) for 15 days. In other embodiments, the antibody is directed against human TNF-α. In other embodiments, the antibody is antibody A.

[0108] Route of administration

[0109] The antibody preparations of the present invention can be administered by a variety of methods known in the art. In some embodiments, they are administered by subcutaneous injection. In other embodiments, they are administered by intravenous injection, intravenous infusion or perfusion. It is known to those skilled in the art that the route of administration varies depending on the desired results.

[0110] In some embodiments, the antibody preparation is administered to the patient by subcutaneous injection. In some embodiments, the dosage of the antibody is 20 mg / time-100 mg / time; in some embodiments, the dosage is about 50 mg / time. The frequency of administration of the antibody is 1 week / time-6 weeks / time; in some embodiments, the frequency of administration is 4 weeks / time. In some embodiments, the antibody preparation is administered to the patient by subcutaneous injection, once every 4 weeks or monthly, each time about 50 mg of anti-TNF-α antibody.

[0111] In some embodiments, the patient is a patient suffering from psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, or the like.

[0112] In some embodiments, the patient is a patient suffering from ulcerative colitis, and the dosage is about 200 mg for the first administration, about 100 mg for the second week, and then about 100 mg of the anti-TNF-α antibody once every 4 weeks.

[0113] TNF-α-related diseases

[0114] The term "TNF-α-related disease" as used herein includes the following diseases: the presence of TNF-α in subjects suffering from the disease has been demonstrated or TNF-α is suspected to be the cause of the pathophysiology of the disease or a factor that exacerbates the disease. TNF-α-related diseases include, but are not limited to, chronic inflammatory diseases, such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, and ulcerative colitis.

[0115] Example

[0116] Example 1: Antibody A protein amino acid sequence and its expression and purification

[0117] 1. Amino acid sequence of antibody A protein

[0118] The anti-TNF-α human antibody (antibody A) used in the preparation of the present invention is a recombinant anti-tumor necrosis factor α fully human monoclonal antibody, which belongs to IgG1 antibody, has a molecular weight of about 150kDa, and is composed of 2 IgG1 heavy chains and 2 κ light chains. Each heavy chain contains 456 amino acids, a molecular weight of 51kDa, and its heavy chain amino acid sequence is shown in SEQ ID NO.1 in the sequence table; each light chain contains 215 amino acids, a molecular weight of 24kDa, and the light chain amino acid sequence is shown in SEQ ID NO.2 in the sequence table. It can be expressed in CHO cells by recombinant DNA technology and purified by a series of standard chromatography steps.

[0119] 2. Expression and purification of antibody A protein

[0120] First, refer to the molecular biology molecular cloning method (such as the method taught in "Molecular Biology of the Gene" published in 2014) to construct an expression vector containing the antibody gene, and then refer to the method of Wood et al., J Immunol. 145: 3011 (1990), etc., and use CHO-K1 cells (ATCCCL61) as host cells to express antibody A. The construction process of the stable cell line is described as follows: the host cells are suspended and grown in CD CHOAGT TM Culture medium (Gibco, CA, 12490017), centrifuge the host cells in the logarithmic growth phase, and resuspend them in fresh CD CHOAGT TMCulture medium, take 0.8 ml of the above cell suspension and add it to the electroporation cup, then add 40 μg of two plasmids containing light chain and heavy chain coding genes that have been double-digested (pre-constructed expression vectors containing antibody genes, the plasmids used are pFUSE-CLIg and pFUSE-CHIg, Invivogen, catalog number pfuse2-hclk and pfuse-hchg1), and mix the cells and plasmids evenly. Transform using Bio-rad electroporator (Bio-rad, Gene PulserXcell). Immediately resuspend the electroporated cells in CD CHOAGT preheated at 37°C TM 0.1 ml of culture medium was added to each well of a 96-well plate. After 2-3 days, an equal amount of screening medium (CD CHOAGT TM Culture medium + 50 μM methionine iminosulfone (MSX, purchased from Sigma-Aldrich, 76078)). The expression level of antibodies in the supernatant of 96-well cell culture was determined using a multifunctional microplate reader (Molecular Devices, Spectramax M4). The clones with higher expression levels were transferred from the 96-well plate to a 24-well plate containing screening culture medium for culture. After the cells grew for 7 days, the cells were transferred to a 6-well plate containing screening culture medium for culture, the antibody production of the cells was determined, and the clones with high expression were transferred to a shake flask. 5-8 clones with the highest expression were screened for subcloning and the above screening process was repeated to finally obtain a strain of engineered cells with the highest expression. The cells were fermented and cultured using a bioreactor. The culture medium was separated from the cells by low-speed centrifugation, and the supernatant was further clarified by high-speed centrifugation.

[0121] Use protein purification liquid chromatography system (General Electric Medical, AKTAPROCESS10MM) to carry out preliminary purification with affinity chromatography (gel medium, MabSelectSuRe LX (GE)). This purification step can achieve the desired purpose, effectively capture the target protein while effectively removing most of the foreign protein, use PowerPac Basic (BIO-RAD) to adopt " Practical Molecular Biology Operation Guide ", People's Medical Publishing House, the method introduced in 2003 publication is carried out SDS-PAGE detection target protein purity, purity reaches more than 90%.

[0122] After preliminary purification, further separation and purification was performed using a protein purification liquid chromatography system (same as above) with anion (Capto Q medium, GE) and cation (Capto S ImpAct medium, GE) exchange chromatography to further remove impurities such as endotoxin, HCP, and DNA. After size exclusion chromatography (SEC) detection (same as above), the SEC monomer content was above 99%.

[0123] Example 2: Screening study of surfactant concentration in antibody preparations

[0124] Antibody A prepared in Example 1 was used to prepare antibody A preparation samples containing different surfactant (polysorbate 80) levels:

[0125] B1: does not contain surfactant;

[0126] B2: Contains 0.1 mg / ml polysorbate 80;

[0127] B3: Contains 0.2 mg / ml polysorbate 80;

[0128] B4: Contains 0.4 mg / ml polysorbate 80;

[0129] Antibody A, stabilizer, buffer and solvent and their concentrations are shown in Table 1 below (the surfactant in Table 1 is 0.2 mg / ml, which is taken as an example).

[0130] Table 1 List of ingredients of Antibody A preparation

[0131]

[0132] The four preparations B1-B4 were diluted with 0.9% sodium chloride solution at a ratio of 1:200, and gently shaken to mix thoroughly. After standing for 2 hours, insoluble particles were detected using a liquid particle counter (Beckman Coulter, HIAC 9703+) according to the method recommended by the manufacturer. The test results are shown in Table 2.

[0133] Table 2 Test results of the number of insoluble particles of antibody A preparations containing different surfactant concentrations after dilution

[0134]

[0135] As shown in the table above, the number of particles of the antibody preparation (B1) without surfactant is significantly higher than that of other preparations (B2-B4), and the number of particles of the antibody preparation (B2-B4) with surfactant added is significantly reduced. The amount of surfactant added also affects the number of particles. Among the preparations B2-B4 with different amounts of surfactant added, the number of particles of the preparations (B3 and B4) containing 0.2 mg / ml and 0.4 mg / ml polysorbate 80, especially the number of particles ≥10 μm, is significantly lower than that of the preparation (B2) containing 0.1 mg / ml polysorbate 80, and the number of particles of the antibody preparation B3 containing 0.2 mg / ml polysorbate 80 is the lowest.

[0136] Example 3: Buffer Screening Test

[0137] Since different buffers have different buffering capacities, in order to find suitable formulation buffers, a variety of formulations containing different buffers were prepared (see Table 3). The other ingredients in the formulation except the buffer and their contents are the same as those shown in Table 1, and the pH of each formulation is 5.5. The storage temperature is 40°C and the time period is 4 weeks. After 4 weeks of storage, each preparation was subjected to size exclusion chromatography (SEC) using a high performance liquid chromatograph (Agilent, 1260 Infinity) using the method disclosed in Zhao J et al. Characterization of the size variants ofarecombinant humanized monoclonal antibody (rhumAb1). Acta Pharmaceutica Sinica, 2016: 1897-1905. The test results are shown in Figure 1 .

[0138] Table 3 Various formulations containing different buffers

[0139]

[0140] like Figure 1 As shown in Table 3, the SEC monomer purity of the five preparations was significantly different. His buffer had the best protective effect on the antibody, followed by Glu and NaAc buffers.

[0141] Example 4: pH screening study of antibody preparations

[0142] pH is a key factor in maintaining antibody stability. Specific antibodies need to be under specific pH conditions to remain stable. Therefore, in order to study under what pH conditions the antibodies of the present invention have better stability, a pH screening test was conducted. Six histidine buffers with different pH values ​​were prepared, covering the range of pH 4.5 to 6.5. The specific test groups are: Sample No. 1, pH 4.5; Sample No. 2, pH 5.0; Sample No. 3, pH 5.5; Sample No. 4, pH 5.8; Sample No. 5, pH 6.0; Sample No. 6, pH 6.5. In addition to the buffer, the other ingredients and contents in the preparation are shown in Table 1.

[0143] The above 6 groups of samples were subjected to a high temperature (40°C) comparative test. Samples taken at time points of 1 week, 2 weeks, 3 weeks, and 4 weeks were subjected to ion exchange chromatography (IEC) detection using a high performance liquid chromatograph (Agilent, 1260 Infinity) using the method disclosed in Harris RJ et al., Identification of multiple sources of charge heterogeneity in a recombinant antibody. Journal of chromatography. B, Biomedical sciences and applications, 2001, 752 (2): 233-245 to detect the antibody stability of each preparation. The test results are shown in Table 4 below.

[0144] Table 4 High temperature comparison test results of samples in different pH ranges

[0145]

[0146] From the IEC-HPLC test data, it can be seen that the IEC-HPLC main peak and acid peak contents of the six groups of samples are significantly different, sample No. 6 is the worst, sample No. 5 is slightly better, and the differences in the other four groups of samples are small, indicating that the antibody preparation of Antibody A has better IEC stability under conditions of pH less than 6.0. At pH 5.5, the main peak of the Antibody A sample is the highest and the acid peak is the lowest, indicating that the stability of the Antibody A preparation is best at pH 5.5.

[0147] Example 5: Stabilizer screening test

[0148] In order to screen the stabilizer for the antibody preparation of the present invention, antibody preparations containing different stabilizers were prepared. The stabilizers used and their contents are shown in Table 5, and the other ingredients in the preparations except the stabilizers are the same as those shown in Table 1. Each preparation was placed in a light incubator at 25°C and irradiated (light intensity 4000lx) for 2 weeks. After 0 days, 7 days, and 2 weeks of illumination, each preparation was subjected to ion exchange chromatography (IEC) detection to detect the antibody stability of each preparation, and the test results are shown in Table 6.

[0149] Table 5 Preparations containing different stabilizers

[0150]

[0151] Table 6 Light stability test data of formulation samples containing different stabilizers

[0152]

[0153]

[0154] Those skilled in the art know that the acid peak of an antibody will gradually increase with the extension of storage time, and that a high acid peak will have an adverse effect on the activity of the antibody. The mutual transformation of the acid peak and the main peak is manifested as the change of the acid peak and the main peak.

[0155] In this experiment, as shown in the data of Table 6 above, it can be seen from the IEC acid peak and main peak content data that after 2 weeks of storage, the acid peak of the No. 10 preparation sample has a small increase, and the main peak content decreases the least, indicating that the No. 10 preparation sample has good stability after 2 weeks of storage. This shows that under light conditions, the stabilizer used in the No. 10 preparation, namely trehalose, has a good protective effect on the stability of the preparation sample.

[0156] In addition, after 2 weeks of storage, the main peak content of preparation No. 7 decreased slightly, and the acid peak increased slightly, indicating that the stability of preparation No. 7 was also good after 2 weeks of storage. That is, arginine hydrochloride also has a good protective effect on the stability of the preparation samples. From the data of preparation samples No. 8 and No. 9, it can be seen that glycine and methionine also have a good protective effect on the preparation samples.

[0157] Example 6: Preparation of Antibody A Preparation

[0158] Based on the studies described in Examples 2-5 above, the antibody A preparation of the present invention was prepared using 0.2 mg / ml polysorbate 80 as a surfactant, His buffer as a buffer, and trehalose as a stabilizer, with a pH of 5.5.

[0159] The materials of the antibody A preparation of the present invention include: trehalose, polysorbate 80, histidine, histidine hydrochloride, antibody A and water for injection.

[0160] The pharmaceutical preparation of the present invention is prepared by the following method:

[0161] (1) Weigh out the following ingredients according to the following weights: 850 g of trehalose dihydrate, 2 g of polysorbate 80, 2.8 g of histidine, and 17.2 g of histidine hydrochloride. Prepare 10 L of water for injection.

[0162] (2) The weighed components are dissolved in about 9 L of water for injection (the order of adding trehalose dihydrate, polysorbate 80, histidine, and histidine hydrochloride can be selected at will), thereby preparing a solvent system.

[0163] (3) The solvent system prepared as above was mixed with the antibody A concentrate containing 1000 g of total protein under stirring, and finally quantified to 10 L with water for injection to obtain the initial preparation of the present invention.

[0164] (4) The initial preparation prepared as above is sterilized by passing through a hydrophilic polyvinylidene fluoride or polyethersulfone filter membrane with a pore size of 0.22 μm. The filter membrane can filter the preparation into a sterile container.

[0165] (5) After sterilization, the formulation is packaged for use, for example in a pre-filled syringe.

[0166] The volumes of the formulations prepared as above and the weights of the materials used may be proportionally scaled up or down, for example, 8L, 7L, 6L, and 5L formulations include 80%, 70%, 60%, and 50% of the listed weights, respectively.

[0167] Example 7: Stability study of the antibody A preparation of the present invention at high temperature (40°C)

[0168] The stability of the antibody A preparation prepared in Example 6 at high temperature was further tested. The antibody A preparation prepared as above was stored at high temperature (40° C.) for 15 days, and then capillary electrophoresis (CE) was performed using a capillary electrophoresis instrument (Agilent, CE 7100) using the method disclosed in Chen G et al., Characterization of glycoprotein biopharmaceutical products by Caliper LC90 CE-SDS gel technology. Methods Mol Biol, 2013, 988 (988): 199-209. The results of the test are shown in FIG. Figure 2 .

[0169] Depend on Figure 2 As shown, when the antibody A preparation was stored at high temperature (40°C) for 15 days, the main peak of antibody CE decreased slowly, from 98.11% to 96.79%, which was higher than 95%, indicating that the stability of the antibody in the antibody A preparation was good, indicating that the preparation of the present invention has an excellent protective effect on the antibody A therein.

[0170] Example 8: Freeze-thaw stability study of the antibody A preparation of the present invention

[0171] Referring to the method described in Example 6 above, antibody A preparations with low antibody concentration (LC, 5 mg / mL), medium antibody concentration (MC, 50 mg / mL), and high antibody concentration (HC, 100 mg / mL) were prepared respectively. Each preparation was stored in a -80°C refrigerator, then taken out and placed at a constant temperature of 25°C to dissolve, and the freeze-thaw was repeated 5 times to examine the stability of the samples after 5 freeze-thaw cycles. After each preparation was frozen and thawed 5 times, 1×10 5, 1×10 6 and 1×10 6 The ELISA test was performed using a multifunctional microplate reader (Molecular Devices, Spectramax M4) using the method disclosed in Wang Yuxiao et al., "Separation, purification and identification of anti-TNF-α human single-chain antibody". Journal of Immunology, 2008 (6): 700-702. Five wells were added to each dilution of the antibody, and the recovery rate and coefficient of variation (CV) of the test are listed in Table 7.

[0172] Table 7 Freeze-thaw test results of antibody A preparations with different antibody concentrations

[0173]

[0174] In addition, SEC detection was performed on each dilution of the preparations with different antibody concentrations using a high performance liquid chromatograph (Agilent, 1260 Infinity) using the method disclosed by Zhao J et al. (supra). The results are shown in Figure 3 .

[0175] The results showed that after five freeze-thaw cycles, the antibody preparations with high, medium and low antibody A concentrations of the present invention had a concentration recovery rate ranging from 101.4% to 107.3%, a coefficient of variation ranging from 6.1% to 11.9%, and SEC purity above 98%. This indicates that the antibody preparations with different antibody concentrations of the present invention have stable quality after five repeated freeze-thaw cycles, and the preparations have a good protective effect on antibody A.

[0176] Example 9: Microbiological Studies

[0177] Microbiological studies of the antibody A preparation of the present invention were further conducted. The antibody A preparation was prepared as described in Example 6 above, and a microbiological study was performed on it to determine whether the preparation can support microbial growth. Microorganisms (Escherichia coli (CICC-10003), Staphylococcus aureus (CICC-10306), Pseudomonas aeruginosa (CICC-10351), Bacillus cereus (CICC-10352)) were inoculated into the above sterile preparations, respectively, with an inoculation amount of 100 cfu / ml, and after being stored at room temperature of 20-25°C for 14 days, the overall microbial growth of the preparations inoculated with each microorganism was detected in the inoculated container. The evaluation indicators mainly include the number of microorganisms under a microscope (Carl Zeiss, Axiovert 25662798) and the change in turbidity (using a turbidity meter (HACH, 2100AN 4700100)) (the test results are shown in Table 8 below), wherein the unchanged turbidity indicates that there is no microbial growth. The data shown in Table 8 below indicate that after storage at room temperature 20-25°C for 14 days, no microorganisms were observed under a microscope and the turbidity of the preparation did not change, thus the preparation of the present invention does not support microbial growth.

[0178] Table 8 Microbiological test results of antibody A preparation

[0179] Test indicators Microbial count cfu / ml Turbidity / NTU Escherichia coli 0 Turbidity unchanged Staphylococcus aureus 0 Turbidity unchanged Bacillus cereus 0 Turbidity unchanged Pseudomonas aeruginosa 0 Turbidity unchanged

[0180] Example 10: Comparative study on the pharmacodynamics of antibody A preparation and Simponi

[0181] The antibody A preparation prepared as above was subjected to in vitro and in vivo pharmacodynamic studies in animal models. The in vitro test mainly carried out the following pharmacodynamic tests: binding ability with TNF-α, affinity with TNF-α, and inhibition of TNF-α biological activity.

[0182] The affinity of the antibody A of the present invention to TNF-α was verified. The specific experimental method is as follows: First, the antibody A preparation prepared in Example 6 above was diluted to 2 μg / ml with PBS; TNF-α (Beijing Sino Biological Technology Co., Ltd., 10602-HNAE) was diluted to 20 nM with PBS, and a 2-fold gradient dilution was performed to obtain 20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 nM TNF-α dilutions. BIAcore (GE Healthcare, T200) was used for detection with Protein A chip (GE Healthcare, 29127556). 2 μg / ml antibody A preparation was passed through the experimental flow path (Fc2, Fc4) at a flow rate of 10 μl / min, and the capture was performed for 10 s to make the capture amount about 200 RU; then the flow rate was adjusted to 30 μl / min, and TNF-α dilutions of different concentrations were passed in sequence, passing through the experimental flow path (Fc2, Fc4) and the reference flow path (Fc1, Fc3) surface respectively, with a binding time of 120 s and a dissociation time of 600 s. Finally, Glycine 1.5 (Shanghai Aladdin Biochemical Technology Co., Ltd., A110752) was introduced for 60 s to regenerate the chip and enter the next cycle. The test results were analyzed using the data analysis software Evaluation Software 3.1. The sensor signals collected from the sample test flow path were double-deducted by the reference flow path and the sample blank, and a 1:1 model was used for kinetic fitting to obtain the kinetic parameters of each batch of samples with TNF-α (ka: binding rate; kd: dissociation rate; kD: binding dissociation equilibrium constant). The same experiment was performed with commercially available Simponi for comparison. The results showed that the affinity constant of the antibody A preparation prepared in the present invention for binding to TNF-α was about 60pM (56.6pM to 67.4pM for the antibody A preparation and 50.9pM to 72.1pM for Simponi), indicating that the anti-TNF-α antibody preparation of the present invention has a strong affinity for TNF-α.

[0183] The antibody A standard (hereinafter referred to as standard S, standard S has a protein content of 110.4 mg / ml and a protein content of 1.980 × 10 5 U / mg activity of antibody A) and Simponi were measured using the same method as above, and the relative affinity of antibody A of the present invention and the commercial Simponi antibody relative to the standard S was further calculated. The results showed that the relative affinity of antibody A of the present invention and the antibody in Simponi for binding to TNF-α was basically the same, as shown in Figure 4 shown.

[0184] In addition, the antibody A preparation prepared by the present invention was verified for its ability to bind to TNF-α (for the test method, see Wang Yuxiao et al., Isolation, Purification and Identification of Human Single-Chain Antibodies against TNF-α. Journal of Immunology, 2008 (6): 700-702) and its inhibitory effect on the biological activity of TNF-α (for the test method, see Trost LC et al., A cytotoxicity assay for tumor necrosis factor employing a multiwell fluorescence scanner. Analytical Biochemistry, 1994, 220 (1): 149-153). The results showed that the EC50 of the antibody A preparation prepared by the present invention for binding to TNF-α was about 8 ng / ml, and the EC50 of its inhibitory effect on the biological activity of TNF-α was about 6 ng / ml.

[0185] The relative binding capacity and relative activity inhibition capacity of the antibody A preparation of the present invention relative to the standard substance S with TNF-α were further calculated. The relative binding capacity and relative activity inhibition capacity of the antibody of the commercially available Simponi relative to the standard substance S with TNF-α were measured using the same method as above. The results are shown in Figure 5 and Figure 6 .like Figure 5 and Figure 6 As shown, the binding ability of the antibody A preparation of the present invention and Simponi to TNF-α, as well as the inhibitory effect of the antibody A preparation of the present invention and Simponi on the biological activity of TNF-α are basically the same.

[0186] The in vivo pharmacodynamic evaluation of the antibody A prepared by the present invention was carried out in a human TNF transgenic spontaneous arthritis mouse model (Tg197, Biomedcode Hellas SA). The injection method adopted was intraperitoneal injection. The transgenic mice were divided into 5 groups, each with 8 mice, and injected with 1 mg / kg of Simponi (G1), saline (G2), 1 mg / kg of antibody A (G3), 5 mg / kg of antibody A (G4) and 5 mg / kg of Simponi (G5), respectively. Injections began from the third week and were injected twice a week until the tenth week. A control group of transgenic mice (4 mice) was added, and this group was sacrificed before the first medication to indicate the initial histopathological state. All experimental group animals were sacrificed in the tenth week, and the ankle joints were collected for pathological evaluation. The scoring system used for the degree of arthritis lesions in vivo and the pathological scoring system used for the histopathological evaluation of the ankle joints are shown below.

[0187] In vivo arthritis lesion severity scoring system:

[0188]

[0189]

[0190] Ankle Histopathology Assessment System:

[0191]

[0192] The experimental results are as follows Fig. 7A , Figure 7B As shown, it is shown that the antibody A of the present invention can inhibit the onset of arthritis in mice within the dosage range of 1 mg / kg and 5 mg / kg, indicating that the antibody A of the present invention has a therapeutic effect on the tg197 arthritis model.

[0193] Example 11: Comparison of molecular weight and glycosylation between the antibody A of the present invention and the commercially available antibody of Simponi

[0194] The intact molecular weight and desugared molecular weight of the antibody A of the present invention and Simponi were detected by a high-resolution mass spectrometer (Waters, Xevo G2-S QTof) according to the method recommended by the manufacturer.

[0195] Antibody A of the present invention and the antibody of simpurin were desugared. The antibody was diluted to about 1 mg / ml with 50 mM NH4HCO3 (pH 8.0), and N-glycosidase PNGaseF (Sigma, catalog number P7367-50UN) was added and reacted at 37°C for about 20 hours. The molecular weight of the antibody A of the present invention and the antibody of simpurin after desugaring was then detected. The results are shown in FIG. Fig. 8A and Figure 8B shown.

[0196] Depend on Fig. 8A The results of the intact molecular weight comparison between the antibody A of the present invention (i.e., the anti-TNF-α antibody in the figure) and the simponi antibody show that, unlike the antibody A of the present invention, simponi has several peaks at molecular weights of 150178 Da and several larger molecular weights, and these peaks disappear after the desugaring treatment ( Figure 8B ). It can be concluded that the difference in molecular weight between the antibody A of the present invention and the antibody of Simponi comes from the difference in their NGNA glycosylation levels. The antibody A of the present invention has a lower NGNA glycosylation level than the antibody of Simponi, resulting in a difference in molecular weight between the two.

[0197] The content of NGNA in Antibody A and Sinimponi was detected (Spichtig V, Michaud J, Austin S. Determination of sialic acids in milks and milk-based products [J]. Analytical Biochemistry, 2010, 405 (1): 28-40.), and the average of three batches of samples was measured. The results are shown in Table 9, and the unit is mol / mol antibody, that is, the molar amount of NGNA in each mole of antibody.

[0198] Table 9 Antibody A and NGNA content in Simponi

[0199] Sample 1 Sample 2 Sample 3 Mean Antibody A 0.0043 0.0043 0.0039 0.0042 Simpney 0.29 0.35 0.29 0.31

[0200] For the amino acid sequences of antibody A and simponi antibody of the present invention, glycosylation modification mainly produces NGNA (N-glycolylneuraminic acid). NGNA glycosylation modification of TNF-α will cause the antibody to be immunogenic to the human body after entering the human body, resulting in adverse reactions. Compared with simponi antibody, antibody A of the present invention has a lower level of NGNA glycosylation modification, so the risk of adverse reactions in the human body caused by NGNA is lower.

[0201] Example 12: Comparison of Fc-terminal activity of antibody A of the present invention and commercially available antibody against TNF-α

[0202] The binding activity of the Fc end of an antibody to various receptors on the cell surface can affect various properties of the antibody. For example, the binding of the Fc end of an antibody to FcRn on the cell surface can affect the half-life of the antibody in the human body, and the binding of the Fc end of an antibody to FcγRIIIa on the cell surface can affect the ADCC (antibody-dependent cell-mediated cytotoxicity) of the antibody.

[0203] This experiment verifies the affinity of antibody A of the present invention and the antibody of simpuny to FcRn. The specific experimental method is as follows: FcRn-biotin (ACRO Biosystems, Cat#FCM-H82W4) was diluted to 1 μg / ml with PBS. SABiosensor (Fertebio, Cat#18-5019) was taken to solidify the receptor protein in the FcRn-biotin solution until the signal was about 1.2 nm. Antibody A of the present invention and the antibody of simpuny were diluted to 200 nM with PBS, and then a 2-fold gradient dilution was performed until 3.125 nM. PBS, drug diluent, 1M MgCl2 (pH 8.3) solution, and PBST were added to the corresponding columns of the 96-well plate in sequence, and the following steps were run on the Octet Platform instrument (Fertebio, Octet QKe) using the SABiosensor with immobilized FcRn: baseline: baseline detection in PBS for 60s; association: binding in drug diluent (200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM) and sample blank (PBS) for 60s; dissociation: dissociation in PBS for 60s; regeneration: regeneration in 1M MgCl2 (pH 8.3) solution for 5s; neutralization: neutralization in PBST for 5s. The regeneration and neutralization cycles were performed 3 times. The data were processed and analyzed using DataAnalysis 8.2 (Fertebio), and the sample data were fitted after the sample blank (PBS) signal was deducted to obtain the affinity constant KD. The results showed that the affinity constant of the antibody A prepared in the present invention for binding to FcRn was about 7 nM (6.35 nM to 7.63 nM for antibody A and 6.09 nM to 8.67 nM for simponi).

[0204] The relative affinity of antibody A of the present invention to FcRn binding relative to standard S was calculated. The above experiment was performed using Ximponi, and the relative affinity of Ximponi's antibody to FcRn binding relative to standard S was calculated. The relative affinity of antibody A of the present invention and Ximponi's antibody to FcRn binding was basically the same, and the results are shown in Fig. 9 .

[0205] In addition, the affinity of the antibody A of the present invention and the antibody of Simponi to FcγRIIIa was also verified. FcγRIIIa has two forms, FcγRIIIa (F158) and FcγRIIIa (V158), both of which were experimentally tested.

[0206] The specific experimental method for binding to FcγRIIIa (F158) is as follows: FcγRIIIa (F158)-biotin (ACROBiosystems, Cat#CDA-H82E8) is diluted to 1 μg / ml. SABiosensor (Fertebio, Cat#18-5019) is solidified in the FcγRIIIa (F158)-biotin dilution solution until the signal is about 0.6 nm. Antibody A and Simponi of the present invention are diluted to 500 nM with PBS, and then 2-fold gradient dilution is performed until 7.812 nM. PBS, drug dilution, 1M MgCl2 (pH 8.3) solution, and PBST were added to the corresponding columns of the 96-well plate in sequence, and the following steps were run on the Octet Platform instrument (Fertebio, Octet QKe): baseline: baseline detection in PBS for 60 s; association: binding in drug gradient dilution (500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.62nM, 7.812nM) and sample blank (PBS) for 60 s; dissociation: dissociation in PBS for 60 s; regeneration: regeneration in 1M MgCl2 (pH 8.3) solution for 5 s; neutralization: neutralization in PBST for 5 s. Regeneration and neutralization cycles were performed 3 times. Data were processed and analyzed using DataAnalysis 8.2, and the sample data were fitted after deducting the sample blank (PBS) signal to obtain the affinity constant KD. The results showed that the affinity constant of the antibody A prepared in the present invention for binding to FcγRIIIa (F158) was about 100 nM (110 nM to 116 nM for antibody A and 93 nM to 111 nM for simponi).

[0207] The relative affinity of the antibody A of the present invention to the standard substance S and FcγRIIIa (F158) was calculated. The above experiment was performed using Simponi, and the relative affinity of the antibody of Simponi to the standard substance S and FcγRIIIa (F158) was calculated. The relative affinity of the antibody A of the present invention and the antibody of Simponi to FcγRIIIa (F158) was basically the same, and the results are shown in Fig.10 .

[0208] The specific experimental method for binding to FcγRIIIa (V158) is as follows: FcγR IIIa (V158)-biotin (ACRO Biosystems, Cat#CDA-H82E9) is diluted to 1 μg / ml. SABiosensor (Fertebio, Cat#18-5019) is immobilized in the FcγR IIIa (V158)-biotin dilution solution until the signal is about 0.6 nm. Antibody A and Simponi of the present invention are diluted to 500 nM with PBS, and then 2-fold gradient dilution is performed until 7.812 nM. PBS, drug dilution, 1M MgCl2 (pH8.4), and PBST were added to the corresponding columns of the 96-well plate in sequence, and the following steps were run on the Octet Platform instrument (Fertebio, Octet QKe): baseline: baseline detection in PBS for 60s; association: binding in drug gradient dilution (500nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.62nM, 7.812nM) and sample blank (PBS) for 60s; association: dissociation in PBS for 60s; regeneration: regeneration in 1M MgCl2 (pH 8.4) for 5s; neutralization: neutralization in PBST for 5s. Regeneration and neutralization cycles were performed 3 times. Data Analysis 8.2 was used to process and analyze the data, and the sample data was fitted after the sample blank (PBS) signal was deducted to obtain the affinity constant KD. The results showed that the affinity constant of the antibody A prepared in the present invention for binding to FcγRIIIa (V158) was about 30 nM (29.2 nM to 40.8 nM for antibody A and 20.1 nM to 42.2 nM for simponi).

[0209] The relative affinity of the antibody A of the present invention to the standard substance S and FcγRIIIa (V158) was calculated. The above experiment was performed using Simponi, and the relative affinity of the antibody of Simponi to FcγRIIIa (V158) was calculated. The relative affinity of the antibody A of the present invention and Simponi to FcγRIIIa (V158) was basically the same, and the results are shown in Fig.11 .

[0210] Example 13: Comparison of the pharmacokinetics of the antibody A of the present invention and the commercially available anti-TNF-α antibody Simponi

[0211] The purpose of this example is to study the pharmacokinetics of the antibody A of the present invention and provide a reference for future rational clinical drug use. At the same time, the pharmacokinetics of the antibody A of the present invention and the commercially available anti-TNF-α antibody Simponi were compared.

[0212] The research plan is as follows:

[0213] A total of 6 groups of cynomolgus monkeys (purchased from Yongfu County Xingui Wildlife Breeding Co., Ltd., 3 to 4.5 years old): low-dose group of antibody A of the present invention, high-dose group of antibody A of the present invention, low-dose group of Simponi (Europe, purchased from Janssen Biologics BV), high-dose group of Simponi (Europe), low-dose group of Simponi (USA, purchased from Janssen Biologics BV), high-dose group of Simponi (USA), 6 animals in each group, half male and half female, 3 mg / kg for low-dose group, and 10 mg / kg for high-dose group. Subcutaneous injection was used for administration, and whole blood was collected at 1h, 6h, 24h, 48h, 3d, 4d, 5d, 7d, and 9d after administration, plasma was separated, and the concentration of the drug in each sample was analyzed.

[0214] Method for determining the antibody content in plasma samples: First, the TNF-α protein (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., item number 10602-HNAE) that can specifically bind to the anti-TNF-α antibody segment is coated on the ELISA plate to capture the antibody in the biological sample, and then the enzyme-labeled goat anti-human antibody (IgG-HRP, purchased from SIGMA, item number A7164) is added, and incubated at room temperature for about 1 hour to allow the enzyme-labeled goat anti-human antibody to bind to the captured anti-TNF-α antibody, and finally the color developing solution (purchased from BD Biosciences, item number 555214) is added, and the color is left at room temperature for 15 minutes. After terminating the reaction with 2mol / L sulfuric acid solution, the OD value of each well is read at 450nm using Spectramax M4 (Molecular Devices). The absorbance of the antibody A standard product S of the present invention and the concentration of the standard sample are fitted by four parameters to obtain a standard curve, and the obtained standard curve is used to quantify the antibody concentration of the sample to be tested measured on the same ELISA plate. The pharmacokinetic parameters of the drug were calculated using WinNonLin6.4 non-compartmental model (NCA), and the average plasma drug concentration-time curve was drawn. The results are shown in FIG12 .

[0215] The results showed that at a dose of 3 mg / kg-10 mg / kg, the pharmacokinetic characteristics of the antibody A of the present invention and Simponi in cynomolgus monkeys were similar.

[0216] Example 14: Stability study of the antibody A preparation of the present invention

[0217] The long-term stability of the antibody A preparation prepared in Example 6 at 5°C±3°C was further investigated. The antibody A preparation prepared as above was stored at 5°C±3°C in the dark for 24 months, and the stability of the antibody A was tested at 0, 3, 6, 9, 12, 18, and 24 months, respectively. The results are shown in Table 10.

[0218] From the results in Table 10, it can be seen that the anti-TNF-α antibody was stored at 5±3°C in the dark for 24 months, and the quality attributes such as SEC, IEC, and CE only fluctuated slightly, indicating that the anti-TNF-α antibody had good stability, indicating that the anti-TNF-α antibody can remain stable for at least 24 months in the anti-TNF-α antibody preparation of the present invention.

[0219] Table 10 Long-term stability results of antibody A formulation at 5°C ± 3°C

[0220] Quality attributes 0 month March June September December 18 months 24 months pH 5.4 5.6 5.5 5.5 5.4 5.5 5.5 SEC-HPLC monomer content (%) 99.8 99.5 99.4 99.4 99.4 99.3 99.3 SEC-HPLC aggregate content (%) 0.2 0.4 0.5 0.5 0.5 0.6 0.6 IEC-HPLC acid peak content (%) 23.8 23.9 21.9 19.3 18.6 18.4 19.5 IEC-HPLC main peak content (%) 53.8 55.1 56.8 56.9 56.2 57.1 54.4 IEC-HPLC alkali peak content (%) 22.4 21.0 21.3 23.8 25.2 24.5 26.1 Non-reduced CE-SDS monomer content (%) 98.3 97.7 98.3 98.3 98.1 98.2 97.8 Protein content (mg / ml) 103.5 102.0 99.3 100.3 99.3 98.4 99.3 Relative biological activity (%) 100 98 101 99 94 93 101 Relative binding activity (%) 99 104 101 102 105 103 92

Claims

1. An anti-TNF-α antibody preparation, characterized in that The preparation comprises an anti-TNF-α antibody and a pharmaceutically acceptable carrier, wherein the anti-TNF-α antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO.1 and a light chain having an amino acid sequence as shown in SEQ ID NO.2, and the pharmaceutically acceptable carrier comprises a stabilizer, a surfactant, and a buffer, wherein the stabilizer is sugar, arginine hydrochloride, glycine, methionine or a combination thereof.

2. The antibody preparation according to claim 1, wherein the pH of the antibody preparation is 4.5-6.

5.

3. The antibody preparation according to claim 1 or 2, wherein the concentration of the anti-TNF-α antibody is 5-130 mg / ml.

4. The antibody preparation according to any one of claims 1 to 3, wherein the concentration of the stabilizer is 160-265 mM.

5. The antibody preparation according to any one of claims 1 to 4, wherein the surfactant is polysorbate.

6. The antibody preparation according to any one of claims 1 to 5, wherein the concentration of the surfactant is 0.05-0.5 mg / ml.

7. The antibody preparation according to claim 1 or 2, wherein the buffer is one or more of a histidine buffer, a glutamic acid buffer, a sodium acetate buffer, a succinic acid buffer and a citrate buffer.

8. The antibody preparation according to any one of claims 1 to 7, wherein the concentration of the buffer is 5-30 mM.

9. An antibody preparation, comprising: 5-130mg / ml anti-TNF-α antibody, 160-265mM stabilizer, 5-30 mM buffer, and 0.05-0.5mg / ml surfactant, pH is 5.0-6.

0.

10. An antibody preparation, comprising: 100mg / ml anti-TNF-α antibody, 225mM trehalose, 10 mM histidine buffer, and 0.2mg / ml polysorbate 80, The pH is 5.0-5.8, preferably pH 5.

5.

11. An antibody preparation, comprising: 90-110mg / ml anti-TNF-α antibody, 210-240mM stabilizer, 10-20 mM buffer, and 0.1-0.3mg / ml surfactant, pH is 4.5-6.

0.

12. An antibody preparation, comprising: 90-110mg / ml anti-TNF-α antibody, 210-240mM trehalose, 10-20 mM histidine buffer, and 0.1-0.3mg / ml polysorbate 80, pH is 4.5-6.

0.

13. The antibody preparation according to any one of claims 9 to 12, wherein the anti-TNF-α antibody comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO.1 and a light chain having an amino acid sequence as shown in SEQ ID NO.

2.

14. A method for preparing an antibody preparation as claimed in any one of claims 1 to 13, comprising: Weigh stabilizers, surfactants, and buffers; Dispersing the weighed components in a liquid solvent for injection, thereby preparing a solvent system; The solvent system is mixed with the anti-TNF-α antibody.

15. An anti-TNF-α antibody pharmaceutical product, comprising the anti-TNF-α antibody preparation according to any one of claims 1 to 13, preferably the antibody pharmaceutical product further comprises a prefilled container.

16. Use of the antibody preparation according to any one of claims 1 to 13, or the antibody pharmaceutical preparation according to claim 15, in the preparation of a medicament for treating TNF-α related diseases.

17. A method for producing an anti-TNF-α antibody, the anti-TNF-α antibody comprising a heavy chain and a light chain, the heavy chain comprising the amino acid sequence shown in SEQ ID NO.1, the light chain comprising the amino acid sequence shown in SEQ ID NO.2, the method comprising CHO cells containing the nucleic acid or vector encoding the anti-TNF-α antibody are cultured to allow the CHO cells to express the anti-TNF-α antibody.

18. An anti-TNF-α antibody, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO.1, and the light chain comprises the amino acid sequence shown in SEQ ID NO.2, and the molar ratio of the Neu5Gc sialic acid (NGNA) level of the anti-TNF-α antibody to that of the anti-TNF-α antibody does not exceed 0.1.

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