CN119454932A

CN119454932A - Anti-TNF-α antibody preparation and preparation method and use thereof

Info

Publication number: CN119454932A
Application number: CN202410931895.4A
Authority: CN
Inventors: 岳海涛; 林键; 吴用; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2020-02-20
Filing date: 2021-02-19
Publication date: 2025-02-18
Also published as: CN113274349A

Abstract

The present invention belongs to the pharmaceutical field, and provides an anti-TNF-α antibody preparation and a preparation method and application thereof, wherein 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. The antibody preparation of the present invention is highly stable and does not support microbial growth.

Description

Antibody preparation for resisting TNF-alpha, preparation method and application thereof

Technical Field

The invention belongs to the field of pharmacy, and in particular relates to an antibody preparation for resisting tumor necrosis factor alpha (TNF-alpha), a preparation method and application thereof.

Background

Tumor necrosis factor alpha (TNF-alpha, tumor necrosis factor alpha), which is secreted by macrophages and monocytes, is an important pro-inflammatory factor and immune regulator, regulating various physiological processes, such as the induction of adhesion molecule expression, the regulation of lymphocyte formation into lymphoid tissue, and the regulation of body's immune defenses. It has been found that TNF- α is considered beneficial to humans when at low concentrations, such as to enhance immune responses, and that TNF- α is at high concentrations, resulting in inflammatory responses and organ damage, such as in patients with sepsis, where TNF- α is produced in large amounts in a short period of time, resulting in shock in the patient. It was found that TNF- α induces expression of adhesion molecules, such as intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), by binding to Tumor Necrosis Factor Receptor (TNFR) on the surface of downstream effector cells, causes neutrophils to aggregate, regulate proliferation, maturation and activation of neutrophils and macrophages, further stimulates monocytes, vascular endothelial cells, etc. to release other cytokines, such as pro-inflammatory factors interleukin 1 (IL-1) and interleukin 8 (IL-8), causing cascade reaction, and finally causes inflammatory injury (Paul A T,Gohil V M,Bhutani K K.Modulating TNF-alpha signaling with natural products.Drug Discovery Today,2006,11(11):725-32.). of tissues to find TNF- α concentration in blood and joint synovium of patients with a plurality of chronic inflammatory diseases, such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, etc., indicating that TNF- α is closely related to these diseases. In recent years many newly developed biological agents can control the progression of a disease by blocking or down-regulating the activity of TNF-alpha, such as TNF-alpha inhibitors. The main action mechanism of the TNF-alpha inhibitor is that after being specifically bound to the secreted tumor necrosis factor alpha (sTNF-alpha), the inhibitor can block the binding with TNFR and inhibit the biological activity of the inhibitor, and can bind with the transmembrane tumor necrosis factor alpha (tmTNF-alpha) on the TNF-alpha synthetic cells, activate a reverse signaling (REVERSE SIGNALING) pathway to reduce the synthesis amount of the TNF-alpha and even cause apoptosis of the TNF-alpha synthetic cells, thereby relieving the inflammatory symptoms of patients.

Currently, the TNF- α inhibitor drugs on the market are mainly Infliximab (Infliximab, trade name) Etanercept (trade name)) Adalimumab (Adalimumab, trade name)) Golimumab (Golimumab, trade name euphorbia)) And cetuximab (Certolizumab pegol, trade name)). Wherein golimumab is a fully human monoclonal antibody, the obtained lot is a subcutaneous injection preparation, and the golimumab is the first international marketed TNF-alpha inhibitor drug which can be subcutaneously administered once in 4 weeks.

From the original murine antibody to the fully human antibody widely used at present, the immunogenicity of the antibody is greatly reduced. However, mammalian cells can synthesize Neu5Gc sialic acid (NGNA) that is immunogenic to humans, such as when using Sp2/0 cells for antibody production, the antibodies are glycosylated to higher levels by NGNA, which may risk eliciting an immune response in humans.

Thus, there remains a need in the art for anti-TNF- α antibodies and formulations thereof having improved properties.

Disclosure of Invention

In one aspect, the 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 under high concentration of antibody protein and preparation stability (especially excellent antibody protein stability under high temperature, light variation, repeated freeze thawing and other conditions), thereby reducing adverse reaction risk, being difficult for microorganism growth and the like. The formulation comprises an anti-TNF-alpha antibody and a pharmaceutically acceptable carrier.

In some embodiments, the anti-TNF- α antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID No.1 and a light chain having the amino acid sequence shown in SEQ ID No. 2.

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.

In some embodiments, the concentration of the anti-TNF- α antibody is from 5to 130mg/ml, in some embodiments, from 50 to 130mg/ml, in some embodiments, from 90 to 110mg/ml, and in some embodiments, 100mg/ml.

In some embodiments, the concentration of the stabilizing agent is 160-265mM, in some embodiments, the concentration of the stabilizing agent is 210-240mM, and in some embodiments, the concentration of the stabilizing agent is 225mM. In some embodiments, the stabilizing agent is 225mM trehalose.

In some embodiments, the antibody formulation may be in liquid form, powder form, or any other suitable form. Depending on the route of administration, storage conditions, etc., the powder form may be resuspended as a solution prior to use.

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

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 between 0.05 and 0.5mg/ml, in some embodiments, the concentration of the surfactant is between 0.1 and 0.3mg/ml, and in some embodiments, the concentration of the surfactant is 0.2mg/ml. In some embodiments, the surfactant is polysorbate 80 at 0.2mg/ml.

In some embodiments, the antibody formulation has a pH of 4.5 to 6.5, in some embodiments, 4.5 to 6.0, in some embodiments, 5.0 to 5.8, in some embodiments, 5.2 to 5.8, and in some embodiments, 5.5.

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

In some embodiments, the buffer of the antibody formulation is one or more of histidine buffer, glutamic acid buffer, sodium acetate buffer, succinic acid buffer, and citric acid buffer, in some embodiments, the buffer is histidine buffer, glutamic acid buffer, sodium acetate buffer, in some embodiments, the buffer is histidine buffer. In some embodiments, the buffer is at a concentration of 5 to 30mM, in some embodiments, the buffer is at a concentration of 10 to 20mM, and in some embodiments, the buffer is at a concentration of 10mM. In some embodiments, the buffer is 10mM histidine buffer.

The various pharmaceutically acceptable carriers of the antibody formulation may be provided in admixture with the antibody or may be provided separately and mixed prior to use.

In some embodiments, an anti-TNF-alpha antibody formulation comprising solvent water, an anti-TNF-alpha antibody comprising 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 an antibody formulation comprising 5-130mg/ml of said anti-TNF-alpha antibody, 160-265mM of said stabilizer, 0.05-0.5mg/ml of said surfactant, 5-30mM of said buffer, the antibody formulation having a pH of 4.5-6.5 is provided. In some embodiments, the antibody formulation comprises 90-110mg/ml of said anti-TNF-alpha antibody, 210-240mM of said stabilizer, 0.1-0.3mg/ml of said surfactant, 10-20mM of said buffer, and the pH of said antibody formulation is 4.5-6.0. In some embodiments, the antibody formulation comprises 90-110mg/ml of the anti-TNF-alpha antibody, 210-240mM trehalose, 0.1-0.3mg/ml polysorbate 80, 10-20mM histidine buffer, and the pH of the antibody formulation is between 4.5-6.0. In some embodiments, the antibody formulation comprises 100mg/ml of the anti-TNF-alpha antibody, 225mM trehalose, 0.2mg/ml polysorbate 80, 10mM histidine buffer, and the pH of the antibody formulation is between 5.0 and 5.8.

In some embodiments, an anti-TNF-alpha antibody formulation comprising solvent water, an anti-TNF-alpha antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID NO.1 and a light chain having the amino acid sequence shown in SEQ ID NO.2 at a concentration of 100mg/ml, a stabilizer of 85mg/ml trehalose dihydrate, a surfactant of 0.2mg/ml polysorbate 80, the antibody formulation having a pH of pH5.5, and maintained by adding 1.72mg/ml histidine hydrochloride and 0.28mg/ml histidine to the liquid component of the antibody formulation is provided.

In a second aspect, the invention provides a method of preparing an anti-TNF-alpha antibody formulation as hereinbefore described.

In one embodiment, there is provided a method of preparing a liquid anti-TNF- α antibody formulation as described hereinbefore, the method comprising:

weighing the stabilizer, the surfactant and the buffer,

Dispersing the weighed components in a liquid solvent for injection to prepare a solvent system,

The solvent system was mixed with the anti-TNF-alpha antibody under agitation.

In some embodiments, the buffer may be one or more of histidine buffer, glutamic acid buffer, sodium acetate buffer, succinic acid buffer, and citric acid buffer, the stabilizer may be sugar, arginine hydrochloride, glycine, methionine, or a combination thereof, and the surfactant may be polysorbate.

In some embodiments, a method of making an anti-TNF- α antibody formulation is provided, the method comprising:

Weighing trehalose, polysorbate 80, histidine and histidine hydrochloride;

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

Mixing the solvent system with an anti-TNF- α antibody under agitation;

so that the final concentration of each component is respectively 90-110mg/ml of anti-TNF-alpha antibody, 80-90mg/ml of trehalose, 0.1-0.3mg/ml of polysorbate 80, 1-3mg/ml of histidine hydrochloride and 0.2-0.5mg/ml of histidine, and the final pH of the antibody preparation is 5.0-6.0.

In some embodiments, the final concentration of each component is 100mg/ml anti-TNF- α antibody, 85mg/ml trehalose, 0.2mg/ml polysorbate 80, 1.72mg/ml histidine hydrochloride, and 0.28mg/ml histidine.

The method may further comprise the steps of preparing an anti-TNF-alpha antibody formulation and/or sterilizing and packaging the antibody formulation. The antibody preparation prepared is sterilized, for example, by passing it through a filter membrane of hydrophilic polyvinylidene fluoride or polyethersulfone having a pore size of 0.22. Mu.m.

In a third aspect, the invention provides a method of treating a TNF- α related disorder comprising administering to a patient in need thereof an anti-TNF- α antibody formulation of the invention as described hereinbefore. The TNF-alpha related diseases include, but are not limited to, chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, and the like.

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.

In a fourth aspect, the invention provides an antibody pharmaceutical product for the treatment of a TNF- α related disorder, comprising an anti-TNF- α antibody formulation of the invention as hereinbefore described and a container for holding said formulation. The pharmaceutical product may also include instructions for use. The container may be any container conventionally used in the art for preserving medicaments, such as a prefilled container, a prefilled syringe, a vial, an ampoule, a pouch, etc.

In a fifth aspect, the invention provides the use of an anti-TNF-alpha antibody formulation or antibody pharmaceutical preparation of the invention as hereinbefore described in the manufacture of a medicament for the treatment of a TNF-alpha related disease. The TNF-alpha related diseases include, but are not limited to, chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis and the like.

In a sixth aspect, the invention provides an anti-TNF- α antibody comprising a heavy chain comprising an amino acid sequence as shown in SEQ ID No.1 and a light chain comprising an amino acid sequence as shown in SEQ ID No.2, with low levels of Neu5Gc sialic acid (NGNA) and reduced risk of adverse effects, under conditions which ensure that the activity of the antibody is unaffected. In some embodiments, the anti-TNF- α antibody has a Neu5Gc sialic acid (NGNA) level to anti-TNF- α antibody molar ratio of no more than 0.1. 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. In some embodiments, the CHO cell is a CHO-K1 cell. In some embodiments, the CHO-K1 cells are acclimatized to accommodate suspension cultured CHO-K1 cells.

In a seventh aspect, the invention provides a method of making an anti-TNF- α antibody of the invention. In some embodiments, the methods use CHO cell lines as host cells. In some embodiments, the methods comprise culturing CHO cells containing a nucleic acid or vector encoding the anti-TNF- α antibody, such that the CHO cells express the anti-TNF- α antibody. In some embodiments, the method comprises the steps of 1) transfecting a nucleic acid or vector encoding the anti-TNF- α antibody into a CHO cell, 2) culturing the CHO cell such that the CHO cell expresses the anti-TNF- α antibody, and in some embodiments, the anti-TNF- α antibody comprises a heavy chain having the heavy chain amino acid sequence shown in SEQ ID NO.1 and a light chain having the light chain amino acid sequence shown in SEQ ID NO. 2. In some embodiments, the CHO cell is a CHO-K1 cell.

In some embodiments, the method further comprises the step of harvesting the culture broth after stopping the culturing. In some embodiments, the method further comprises purifying the anti-TNF-a antibody after harvesting the culture broth.

In a seventh aspect, the invention provides an anti-TNF- α antibody prepared by the method as above, said antibody having a reduced level of deamidation and/or a reduced level of NGNA glycosylation. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) to anti-TNF- α antibody in the anti-TNF- α antibody is no more than 0.1, in some embodiments, the molar ratio of NGNA to anti-TNF- α antibody in the anti-TNF- α antibody is no more than 0.01, and in some embodiments, the molar ratio of NGNA to anti-TNF- α antibody in the anti-TNF- α antibody is no more than 0.005.

In some embodiments, the CHO cell-expressed anti-TNF- α antibody comprises a heavy chain amino acid sequence as set forth in SEQ ID No.1, a light chain amino acid sequence as set forth in SEQ ID No.2, and substantially coincides (differs by no more than ±20%, or ±10%, or ±5%, or no more than 3 times, 2 times, or 1 times the standard deviation) with euphoria in one or more of relative affinity to TNF- α, binding to FcRn, binding to fcyriiia (F158), binding to fcyriiia (V158), and pharmacokinetic characteristics (e.g., plasma drug concentration) in cynomolgus monkeys. In some embodiments, the anti-TNF- α antibody expressed by the CHO cell is antibody a.

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

The anti-TNF-alpha antibody developed by the invention has low NGNA glycosylation level under the condition of ensuring the activity of the antibody, so that the activity of the antibody is higher and the immunogenicity is lower. The anti-TNF-alpha antibody preparation developed by the invention comprises high concentration of the anti-TNF-alpha antibody protein, has higher activity and lower immunogenicity, and the high concentration antibody protein contained in the preparation is stable, particularly resistant to protein aggregation reaction caused by high temperature, illumination and repeated freeze thawing, and does not support the growth of microorganisms. Accordingly, the anti-TNF- α antibody formulations of the present invention meet the need in the art for anti-TNF- α antibody pharmaceutical formulations having improved properties.

Drawings

FIG. 1 shows a size exclusion chromatography assay of the buffer screening assay for anti-TNF-alpha antibody formulations as tested in example 3.

FIG. 2 shows a graph of the results of capillary electrophoresis detection of stability studies of anti-TNF-alpha antibody formulations of the present invention under high temperature (40 ℃) conditions.

FIG. 3 shows a graph of size exclusion chromatography measurements of freeze-thaw stability studies of anti-TNF-alpha antibody formulations of the present invention.

FIG. 4 shows the relative affinities of the antibody A preparations of the invention and of euphorbia binding to TNF-alpha.

FIG. 5 shows the relative binding capacity of the antibody A preparation and euphoria of the present invention to TNF- α.

FIG. 6 shows the inhibition of TNF- α biological activity by antibody A formulations of the invention and euphorbia.

Figure 7A shows the effect of antibody a of the invention on arthritis score in Tg197 transgenic spontaneous arthritis mouse model.

FIG. 7B shows the effect of antibody A of the invention on the histopathological score of the Tg197 transgenic spontaneous arthritis mouse model.

FIGS. 8A and 8B show a comparison of the molecular weights of antibody A of the present invention and that of euphorbia (FIG. 8A: intact molecular weight; FIG. 8B: desugared molecular weight).

Figure 9 shows the relative affinities of antibodies of the invention for binding to FcRn for antibody a and euphoria.

Fig. 10 shows the relative affinities of antibodies a and euphorbia of the invention for binding to fcγriiia (F158).

FIG. 11 shows the relative affinities of antibodies A and euphorbia of the present invention for binding to FcgammaRIIIa (V158).

FIG. 12A shows a pharmacokinetic comparison of 3mg/kg in cynomolgus monkeys for both antibody A of the invention and for the low dose group of euphorbia.

FIG. 12B shows a pharmacokinetic comparison of 10mg/kg in cynomolgus monkeys for both antibody A of the invention and for the high dose group of euphorbia.

Detailed Description

In order to make the technical contents of the present invention more clearly understood, the following examples are specifically described. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, various equivalents to the specific embodiments of the invention. Such equivalents are also included within the scope of the present invention. All documents and patents cited herein are incorporated by reference. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

The skilled person will appreciate that the weights and/or weight to volume ratios mentioned herein may be converted to molar and/or molarity using the well known molecular weights of the respective components. The weights listed in the examples are for the corresponding volumes. The skilled person will appreciate that the weight may be adjusted proportionally when different volumes of formulation are required.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Definition of the definition

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

"About" or "approximately" refers to a conventional error range of corresponding numerical values as readily known to one of ordinary skill in the relevant art. In some embodiments, references herein to "about" or "approximately" refer to the values recited and ranges thereof of + -10%, + -5%, + -1%, or + -0.1%.

"Comprising" or "comprises" means that the compositions and methods, etc., include the recited elements (e.g., components in the compositions, steps in the methods, etc.), but do not exclude other. When "consisting essentially of" is used to define compositions and methods, it is meant to exclude other elements that have a radical impact on the combination for the intended use, but not elements that do not materially affect the characteristics of the composition or method. "consisting of" means excluding elements not specifically recited. Implementations defined by each of these transitional terms are within the scope of the invention. For example, when the composition is described as comprising 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 invention.

As used herein, "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be an intact antibody and any antigen binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide comprising a molecule comprising at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen. Examples include, but are not limited to, complementarity Determining Regions (CDRs) of a heavy or light chain or ligand-binding portion thereof, heavy or light chain variable regions, heavy or light chain constant regions, framework (FR) regions or any portion thereof, or at least a portion of a binding protein.

As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or slow (reduce) the progression of an undesired physiological change or disorder, such as TNF-a related disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized condition (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, remission (partial or total), whether detectable or undetectable. The persons in need of treatment include those already with the condition or disorder, as well as those prone to develop the condition or disorder, or those in need of prevention of the condition or disorder.

As used herein, "patient" refers to any subject, particularly a mammalian subject, in need of treatment thereof. Mammalian subjects include humans, domestic animals, livestock, zoo animals, sports animals or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc.

As used herein, "a patient in need thereof" includes patients, such as mammalian patients, who would benefit from administration of the antibodies or formulations of the invention.

The term "pharmaceutically acceptable" as used herein refers to a drug listed in the accepted pharmacopoeia that can be used in animals, especially in humans. The "pharmaceutically acceptable carrier" is typically a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any type of formulation aid.

The term "buffer", also referred to in some documents as buffer systems or buffer systems, 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 salts thereof, tris, thomethamine hydrochloride, or phosphate buffers. In addition, amino acids and salts thereof may 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.

The amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system comprising the buffer. In some embodiments, molar concentration is used as a unit of the amount of buffer (or buffer), which refers to the molar concentration of buffer pairs in the buffer system of the buffer (or buffer). For example, when a histidine buffer consisting of histidine and histidine hydrochloride is used as the buffer, the histidine salt buffer (e.g., 10 mM) at a given concentration is the combined concentration of histidine and histidine hydrochloride (e.g., 5mM for histidine hydrochloride; or 1.8mM for histidine and 8.2mM for histidine hydrochloride).

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

The term "surfactant" includes, but is not limited to, polysorbate (e.g., polysorbate 20 and polysorbate 80), poloxamers (e.g., poloxamer 188), triton, sodium Dodecyl Sulfate (SDS), sodium lauryl sulfate, octyl glycoside sodium salt, 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, lauramidopropyl betaine, cocoamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitoamidopropyl betaine (e.g., lauramidopropyl), myristamidopropyl dimethylamine, palmitoamidopropyl dimethylamine or isostearamidopropyl dimethylamine, sodium methyl cocoyl taurate or disodium methyl cocoyl taurate, polyethylene glycols, polypropylene glycols, and copolymers of ethylene with propylene glycol (e.g., pluronics, 68 PF, etc.). In some embodiments, the surfactant is polysorbate 80.

The term "pharmaceutically acceptable" refers to materials approved by a regulatory agency of the country or listed in the pharmacopeia or other generally recognized pharmacopeia as being useful in animals, and more particularly in humans. Furthermore, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid, or the like.

Anti-TNF-alpha antibodies

The present invention provides anti-TNF-alpha antibodies having high affinity for TNF-alpha proteins. The anti-TNF-alpha antibody disclosed by the invention comprises a heavy chain and a light chain, wherein the heavy chain amino acid sequence is shown as SEQ ID NO. 1 or a sequence with 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence SEQ ID NO. 1, and the light chain amino acid sequence is shown as SEQ ID NO. 2 or a sequence with 80%, 85%, 90%, 95%, 98% or 99% identity with the sequence SEQ ID NO. 2.

The sequence of SEQ ID NO. 1 is:

QVQLVESGGGVVQPGRSLRLSCAASGFIFSSYAMHWVRQAPGNGLEWVAFMSYDGSNKKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRGIAAGGNYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

The sequence of SEQ ID NO. 2 is:

EIVLTQSPATLSLSPGERATLSCRASQSVYSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

In some embodiments, the anti-TNF- α antibody is golimumab # Or a biological analog thereof (e.g., BOW100, ONS-3035, or antibody A)).

In some embodiments, the anti-TNF- α antibodies are prepared by culturing CHO cells transfected with a nucleic acid or vector encoding the anti-TNF- α antibody such that the CHO cells express the anti-TNF- α antibody. In some embodiments, the CHO cell is a CHO-K1 cell. In some embodiments, the CHO-K1 cells are CHO-K1 cells adapted to suspension culture. In some embodiments, the vector may be a plasmid vector, a phage vector, a viral vector, a non-viral vector, or a micro-circular DNA. In some embodiments, culturing the CHO cells includes allowing the CHO cells to express anti-TNF-alpha antibodies under suitable conditions and media.

In some embodiments, the step of culturing the CHO cells comprises culturing under suitable conditions and media for a period of time such that the CHO cells express the heavy and light chains of the antibody and form anti-TNF-alpha antibodies. In some embodiments, the method further comprises the step of stopping the culturing and harvesting the culture broth. In some embodiments, the method further comprises purifying the anti-TNF-a antibody after harvesting the culture broth.

In some embodiments, after harvesting the culture broth, the anti-TNF-a antibody is isolated by centrifuging the culture broth. 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, and the anti-TNF- α antibody is purified by centrifugation, and in some embodiments, the purified anti-TNF- α antibody may be about 90% or more, about 92% or more, about 94% or more, about 96% or more, 98% or more, or about 99% or more pure.

Glycosylation is one of the key quality attributes of antibodies. The realization of the drug function of the monoclonal antibody is closely related to the glycosylation, and glycosylation modification can influence the performance of the protein, such as conformation, stability, solubility, pharmacokinetics, activity and immunogenicity. Glycosylation can be classified into N-glycosylation and O-glycosylation according to the modification site of glycosylation. Glycosylation at the N-position is the most common glycosylation in immunoglobulins secreted by animal cells, whereas antibody glycosylation is also predominantly at the N-position, with golimumab, glycosylation occurring mostly at Asn-306. Depending on the fine structure of the glycosylated terminal (length, branching and monosaccharide arrangement), it is also classified into complex, hybrid and high mannose types, which are closely related to cell species and cell culture conditions.

From the original murine antibody to the fully human antibody widely used at present, the immunogenicity of the antibody is greatly reduced. However, mammalian cells can synthesize Neu5Gc sialic acid (NGNA) that is immunogenic to humans, such as when using Sp2/0 cells for antibody production, glycosylation modified NGNA levels are high, which may risk eliciting an immune response in humans.

The present invention provides an anti-TNF-alpha antibody having a low NGNA level, comprising a heavy chain comprising an amino acid sequence as shown in SEQ ID No.1 and a light chain comprising an amino acid sequence as shown in SEQ ID No. 2. In some embodiments, the anti-TNF- α antibody has a Neu5Gc sialic acid (NGNA) level to anti-TNF- α antibody molar ratio of no more than 0.1. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) to anti-TNF- α antibody in 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 to anti-TNF-a antibody in the anti-TNF-a 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.

The invention provides a method for producing an anti-TNF-alpha antibody, which comprises the following steps of preparing nucleic acid or vector for encoding the anti-TNF-alpha antibody, transfecting the nucleic acid or vector for encoding the anti-TNF-alpha antibody into CHO-K1 cells, enabling the CHO-K1 cells to express the anti-TNF-alpha antibody under proper conditions and culture medium, stopping culturing, harvesting culture solution, separating the culture solution by centrifugation, obtaining the anti-TNF-alpha antibody by centrifugation, carrying out primary purification on the anti-TNF-alpha antibody obtained by centrifugation by affinity chromatography, and further purifying the primary purified anti-TNF-alpha antibody by anion and cation exchange chromatography to obtain the purified anti-TNF-alpha antibody. In some embodiments, the invention provides antibody A, antibody A is an anti-TNF-alpha antibody, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1, the amino acid sequence of the light chain is shown as SEQ ID NO.2, and antibody A is produced by the methods described above.

The present invention provides anti-TNF-alpha antibodies prepared by the method described above, which antibodies have a lower NGNA glycosylation level. In some embodiments, the molar ratio of Neu5Gc sialic acid (NGNA) to anti-TNF- α antibody in 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 to anti-TNF-a antibody in the anti-TNF-a 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.

Dosage form

The antibody formulations of the invention may be liquid solutions (e.g., injectable, infusible solutions), or powders. The form depends on the desired mode of administration and the therapeutic application. The formulation is in the form of an injectable, infusible or infusible 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.

Supplementary active compounds may also be incorporated into the pharmaceutical formulations. In some embodiments, an antibody or antigen-binding portion thereof of the invention is co-formulated and/or co-administered with one or more additional therapeutic agents. For example, the additional therapeutic agent may be a DMARD, an NSAID, other antibodies that bind to other targets (e.g., antibodies that bind to other cytokines or to cell surface molecules), cytokines, soluble TNF-alpha receptors, chemicals that inhibit TNF-alpha production and activity, or any combination thereof. Furthermore, the antibodies of the invention may be used in combination with the therapeutic substances described above. Such combination therapy may advantageously employ lower doses of the therapeutic agent administered, thereby avoiding possible side effects, complications or low level patient responses associated with various monotherapy treatments, or increasing efficacy.

In some embodiments, the antibody formulation is an anti-TNF-a antibody formulation. In some embodiments, an anti-TNF- α antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID No.1 and a light chain having the amino acid sequence shown in SEQ ID No. 2. In some embodiments, the concentration of anti-TNF- α antibody is from about 5 to about 130mg/ml, in some embodiments, from about 50 to about 130mg/ml, in some embodiments, from about 90 to about 110mg/ml, and in some embodiments, about 100mg/ml. In some embodiments, the concentration of the anti-TNF- α antibody is about 5mg/ml, about 10mg/ml, about 30mg/ml, about 50mg/ml, about 70mg/ml, about 80mg/ml, about 90mg/ml, about 100mg/ml, about 110mg/ml, about 120mg/ml, about 130mg/ml, or a range between any two concentrations, inclusive.

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 cell expressing the anti-TNF- α antibody is a CHO-K1 cell.

In some embodiments, the molar ratio of NGNA in the anti-TNF-alpha antibody to the anti-TNF-alpha antibody in the formulation is no more than 0.1, in some embodiments, the molar ratio of NGNA in the anti-TNF-alpha antibody to the anti-TNF-alpha antibody is no more than 0.01, and in some embodiments, the molar ratio of NGNA in the anti-TNF-alpha antibody to the anti-TNF-alpha antibody is no more than 0.005.

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 160-265mM, in some embodiments, the concentration of the stabilizer is about 210-240mM, and in some embodiments, the concentration of the stabilizer is about 225mM. In some embodiments, the stabilizing agent is about 225mM trehalose. In some embodiments, the anti-stabilizer is trehalose or sucrose, and the stabilizer is at a concentration of about 160mM, about 180mM, about 200mM, about 210mM, about 225mM, about 240mM, about 250mM, about 265mM, or a range between any two concentrations, inclusive.

In the solid state, trehalose is typically present as trehalose dihydrate, so in some embodiments, the formulation is formulated using trehalose dihydrate, but can also be formulated using corresponding amounts of other forms of trehalose, resulting in a formulation containing the same concentration of trehalose. When the trehalose dihydrate is used herein to describe the amount of trehalose in the formulation, the formulation may contain the stated amount of trehalose dihydrate or a corresponding amount of trehalose or other forms of trehalose or combinations thereof, and vice versa.

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

In some embodiments, the formulation further comprises a surfactant. In some embodiments, the surfactant comprises a nonionic surfactant, such as polysorbate 80. Surfactants reduce aggregation of the antibodies and/or reduce particle formation and/or reduce adsorption in the formulation. In some embodiments, the surfactant is polysorbate, in some embodiments, polysorbate 20 and/or polysorbate 80, in some embodiments, the concentration of the surfactant is about 0.05 to about 0.5mg/ml, in some embodiments, the concentration of the surfactant is about 0.1 to about 0.3mg/ml, and in some embodiments, the concentration of the surfactant is about 0.2mg/ml. In some embodiments, the surfactant is about 0.2mg/ml polysorbate 80. In some embodiments, the concentration of polysorbate 80 is about 0.05mg/ml, about 0.1mg/ml, about 0.15mg/ml, about 0.2mg/ml, about 0.25mg/ml, about 0.3mg/ml, about 0.4mg/ml, about 0.5mg/ml, or a range between any two concentrations, including the endpoints.

In some embodiments, the antibody formulation has a pH of about 4.5 to about 6.5, in some embodiments, about 4.5 to about 6.0, in some embodiments, about 5.0 to about 6.0, in some embodiments, about 5.2 to about 5.8, and in some embodiments, 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 a range between any two acid-base values, inclusive.

In some embodiments, the buffer of the antibody preparation is a histidine buffer, a glutamic acid buffer, a sodium acetate buffer, a succinic acid buffer, or a citric acid buffer, in some embodiments, the buffer is a histidine buffer, a glutamic acid buffer, a sodium acetate buffer, in some embodiments, the buffer is a histidine buffer. In some embodiments, the buffer is at a concentration of about 5mM to about 30mM, in some embodiments, the buffer is at a concentration of about 10mM to about 20mM, and in some embodiments, the buffer is at a concentration of about 10mM. In some embodiments, the buffer is about 10mM histidine buffer. In some embodiments, the concentration of histidine buffer is in the range between about 5mM, about 10mM, about 20mM, about 30mM, or any two concentrations, inclusive.

In some embodiments, the antibody formulation comprises about 5-130mg/ml of anti-TNF-alpha antibody, about 160-265mM stabilizer, about 0.05-0.5mg/ml surfactant, about 5-30mM buffer, and the pH of the antibody formulation is about 4.5-6.5. In some embodiments, the antibody formulation comprises about 90-110mg/ml of anti-TNF-alpha antibody, about 210-240mM stabilizer, about 0.1-0.3mg/ml surfactant, about 10-20mM buffer, and the pH of the antibody formulation is about 4.5-6.0. In some embodiments, the antibody formulation comprises about 90-110mg/ml anti-TNF- α antibody, about 210-240mM trehalose, about 0.1-0.3mg/ml polysorbate 80, about 10-20mM histidine buffer, and the pH of the antibody formulation is about 4.5-6.0. In some embodiments, the antibody formulation comprises about 100mg/ml anti-TNF- α antibody, about 225mM trehalose, about 0.2mg/ml polysorbate 80, about 10mM histidine buffer, and the pH of the antibody formulation is between 5.0 and 5.8. In some embodiments, the antibody formulation comprises about 100mg/ml anti-TNF- α antibody, about 10mM to about 142mM arginine hydrochloride, about 0.2mg/ml polysorbate 80, about 10mM histidine buffer, and the pH of the antibody formulation is between 5.0 and 5.8. In some embodiments, the antibody formulation comprises about 100mg/ml anti-TNF- α antibody, about 333mM glycine, about 0.2mg/ml polysorbate 80, about 10mM histidine buffer, and the pH of the antibody formulation is between 5.0 and 5.8. In some embodiments, the antibody formulation comprises about 100mg/ml anti-TNF-alpha antibody, about 34mM methionine, about 0.2mg/ml polysorbate 80, about 10mM histidine buffer, and the pH of the antibody formulation is between 5.0 and 5.8. In some embodiments, the anti-TNF-alpha antibody is(Golimumab) or a biological analogue thereof (e.g.BOW 100, ONS-3035 or antibody A).

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

In some embodiments, the amount of acidic antibody variant in the anti-TNF-alpha antibody formulation is no more than about 40%, no more than about 35%, no more than about 30%. In some embodiments, the amount of acidic antibody variant in the anti-TNF- α antibody formulation 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, inclusive.

In some embodiments, the anti-TNF- α antibody formulations provided herein have an acid peak content of no more than 30% after 1 week, 2 weeks, 3 weeks, or 4 weeks of high temperature (40 ± 3 ℃).

The present invention provides liquid aqueous pharmaceutical formulations, including antibodies suitable for therapeutic use, primarily for the treatment of disorders caused by TNF- α. The pharmaceutical formulation is convenient to administer and contains high protein concentrations and can be injected subcutaneously. In some embodiments, the pharmaceutical formulation has a stability enhancing effect and enables deamidation of antibodies to be kept low. In other embodiments, the formulations of the invention are stable after at least 5 freeze-thaw cycles. In other embodiments, the formulations of the present invention remain stable after 15 days of high temperature (40 ℃) exposure. In other embodiments, the antibody is directed against human TNF- α. In other embodiments, the antibody is antibody a.

Route of administration

The antibody formulations of the invention may be administered by a variety of methods known in the art. In some embodiments, administration is by subcutaneous injection. In other embodiments, administration is by intravenous injection, intravenous infusion, or infusion. The person skilled in the art knows that the route of administration varies according to the desired result.

In some embodiments, the antibody formulation is administered to the patient by subcutaneous injection. In some embodiments, the antibody is administered at a dose of 20 mg/time to 100 mg/time, and in some embodiments, at a dose of about 50 mg/time. The frequency of administration of the antibody is 1 week/time to 6 weeks/time, and in some embodiments, 4 weeks/time. In some embodiments, the antibody formulation is administered to the patient by subcutaneous injection 1 every 4 weeks or month, about 50mg of anti-TNF-a antibody each time.

In some embodiments, the patient is a patient suffering from psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, and the like.

In some embodiments, the patient is a patient suffering from ulcerative colitis, and the dose administered is about 200mg for the first time, about 100mg for week 2, and then 1 time every 4 weeks, about 100mg of anti-TNF-a antibody each time.

TNF-alpha related diseases

The term "TNF- α related disease" as used herein includes diseases in which the presence of TNF- α in a subject suffering from the disorder has been demonstrated or suspected to be responsible for the pathophysiology of the disorder or to be a factor contributing to the exacerbation of the disorder. TNF- α related disorders include, but are not limited to, chronic inflammatory disorders such as psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis and the like.

Examples

Example 1 amino acid sequence of antibody A protein and expression and purification thereof

1. Amino acid sequence of antibody A protein

The human antibody (antibody A) of the anti-TNF-alpha adopted in the preparation of the invention is a recombinant anti-tumor necrosis factor alpha fully human monoclonal antibody, belongs to an IgG1 antibody, has the molecular weight of about 150kDa and consists of 2 IgG1 heavy chains and 2 kappa light chains. Each heavy chain contains 456 amino acids, the molecular weight is 51kDa, the amino acid sequence of the heavy chain is shown as SEQ ID NO.1 in a sequence table, each light chain contains 215 amino acids, the molecular weight is 24kDa, and the amino acid sequence of the light chain is shown as SEQ ID NO.2 in the sequence table. Can be expressed in CHO cells by recombinant DNA techniques and purified by a series of standard chromatographic steps.

2. Expression and purification of antibody A proteins

An expression vector containing an antibody gene was constructed by referring to a molecular cloning method in molecular biology (for example, a method taught in Molecular Biology ofthe Gene published 2014), and antibody A was expressed by using CHO-K1 cells (ATCCCL 61) as host cells by referring to Wood et al, JImmunol.145:3011 (1990), and the like. The construction of a stable cell line was described by growing host cells in suspension in CD CHOAGT ^TM medium (Gibco, CA, 12490017), centrifuging the host cells in the logarithmic phase, re-suspending them in fresh CD CHOAGT ^TM medium, adding 0.8ml of the above cell suspension into a cuvette, then adding 40. Mu.g of each of the two double digested plasmids containing the light chain and heavy chain encoding genes (pre-constructed expression vectors containing antibody genes, pFUSE-CLIg and pFUSE-CHIg, invivogen, cat. No. pFUSE-hclk and pFUSE-hchg), and mixing the cells with the plasmids uniformly. Transformation was performed using a Bio-rad electrotransformation apparatus (Bio-rad, gene PulserXcell). Immediately after the shock, the cells were resuspended in CD CHOAGT ^TM medium preheated at 37℃and 0.1ml of 96-well plate was added per well, and an equal amount of screening medium (CD CHOAGT ^TM medium+50. Mu.M methionine iminosulfone (MSX, available from Sigma-Aldrich, 76078)) was added after 2-3 days. The expression level of the antibodies in the cell culture supernatants of 96-well plates was determined using a multifunctional microplate reader (Molecular Devices, spectramax M4). Clones with higher expression level are transferred from a 96-well plate to a 24-well plate containing a screening culture medium for culture, after the cells grow for 7 days, the cells are transferred to a 6-well plate containing the screening culture medium for culture, the antibody yield of the cells is measured, and clones with high expression level are transferred to a shake flask. Screening out 5-8 clones with the highest expression level for subcloning, and repeating the screening process to finally obtain 1 engineering cell with the highest expression level. The cells are fermented and cultured by a bioreactor. The cells and medium are separated by low-speed centrifugation of the culture medium, and the supernatant is further clarified by high-speed centrifugation.

Preliminary purification was performed using a protein purification liquid chromatography system (general electric medical treatment, AKTAPR0CESS MM) with affinity chromatography (gel media, mabSelectSuRe LX (GE)). The purification step can achieve the expected purpose, most of the impurity proteins are effectively removed while the target proteins are effectively captured, and the purity of the target proteins is detected by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) by using PowerPac Basic (BIO-RAD) through a method described in practical molecular biology operation guidelines, people health publishers and 2003 publications, wherein the purity is more than 90%.

After preliminary purification, a protein purification liquid chromatography system (the same as above) is used for further separation and purification by adopting anion (Capto Q medium, GE) and cation (Capto S ImpAct medium, GE) exchange chromatography, so that impurities such as endotoxin, HCP, DNA and the like are further removed, and the content of SEC monomers is more than 99 percent through Size Exclusion Chromatography (SEC) detection (the same as above).

Example 2 surfactant concentration screening study in antibody formulations

Antibody a preparation samples containing different surfactant (polysorbate 80) levels were prepared separately using antibody a prepared in example 1:

b1, no surfactant;

b2, containing 0.1mg/ml polysorbate 80;

b3, containing 0.2mg/ml polysorbate 80;

b4, containing 0.4mg/ml polysorbate 80;

the concentrations of antibody A, stabilizer, buffer and solvent are shown in Table 1 below (surfactant 0.2mg/ml in Table 1 is exemplified).

TABLE 1 list of Components of antibody A formulations

The 4 formulations B1-B4 were diluted 1:200 in 0.9% sodium chloride solution, mixed thoroughly with gentle shaking, and after standing for 2h, insoluble particles were detected using a liquid particle counter (Beckman Coulter, HIAC 9703+) according to the manufacturer's recommendations. The detection results are shown in Table 2.

TABLE 2 detection results of the number of insoluble microparticles after dilution of antibody A preparations containing different surfactant concentrations

As shown in the above table, the number of microparticles of the surfactant-free antibody preparation (B1) was significantly higher than that of the other preparations (B2 to B4), and the number of microparticles of the surfactant-added antibody preparation (B2 to B4) was significantly reduced. The amount of surfactant added also has an effect on the number of particles, and among the formulations B2-B4 to which different amounts of surfactant are added, the formulations (B3 and B4) containing 0.2mg/ml and 0.4mg/ml polysorbate 80 have a significantly lower number of particles, in particular ≡10 μm, than the formulation (B2) containing 0.1mg/ml polysorbate 80, and the antibody formulation B3 containing 0.2mg/ml polysorbate 80 has the lowest number of particles.

Example 3 buffer screening assay

Because of the different buffering capacities of the different buffers, in order to find suitable formulation buffers, various formulations containing the different buffers were prepared (see table 3). The other components except the buffer and the content thereof in the formulations were the same as those shown in table 1, and the pH of each formulation was 5.5. The storage temperature was 40 ℃ and the time period was 4 weeks. After 4 weeks of standing, each formulation was subjected to size exclusion chromatography (size-exclusion chromatography, SEC) using a high performance liquid chromatograph (Agilent, 1260 Infinity) using methods as disclosed in Zhao J et al Characterization ofthe size variants ofa recombinant humanized monoclonal antibody(rhumAb1).Acta PharmaceuticaSinica,2016:1897-1905, the results of which are shown in fig. 1.

TABLE 3 multiple formulations containing different buffers

As shown in fig. 1, the SEC monomer purity differences for the 5 formulations shown in table 3 are evident, with His buffer being optimal for antibody protection and Glu, naAc buffer being less protective.

Example 4 pH screening study of antibody formulations

The pH is a key factor in maintaining antibody stability. Specific antibodies need to be at specific pH conditions to remain stable. Therefore, in order to study under which pH conditions the antibodies of the present invention are stable, a pH screening test was performed. 6 histidine buffers with different pH values are prepared, and the pH value ranges from 4.5 to 6.5. The specific test groups are sample No. 1, sample No. 4.5, sample No. 2, sample No. 5.0, sample No. 3, sample No. 5.5, sample No. 4, sample No. 5.8, sample No. 5, sample No. 6.0, sample No. 6.5. The other ingredients and amounts of the formulations, except for the buffer, are shown in table 1.

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

TABLE 4 results of high temperature comparative test on samples at different pH ranges

As can be seen from IEC-HPLC detection data, the IEC-HPLC main peak and acid peak content of the 6-group samples are obviously different, the 6-group sample is worst, the 5-group sample is slightly better, and the other 4-group samples are smaller in difference, so that the antibody preparation of the antibody A has better IEC stability under the condition that the pH is less than 6.0, wherein the main peak of the antibody A is highest at the pH of 5.5, and the acid peak is lowest, so that the stability of the antibody A preparation is best at the pH of 5.5.

Example 5 stabilizer screening test

In order to screen the stabilizers for the antibody preparations of the present invention, antibody preparations containing different stabilizers are prepared. The stabilizers and their contents are shown in table 5, and the other components except for the stabilizers in the formulations are the same as shown in table 1. Each formulation was kept for 2 weeks under irradiation (4000 lx intensity) in a light incubator at 25 ℃. Ion exchange chromatography (ion exchange chromatography, IEC) was performed on each preparation after 0, 7, and 2 weeks of light irradiation, respectively, to examine the antibody stability of each preparation, and the examination results are shown in table 6.

TABLE 5 formulations containing different stabilizers

TABLE 6 light stability test data for samples of formulations containing different stabilizers

Those skilled in the art know that antibody acid peaks will gradually increase with longer shelf life, and that acid peaks that are too high have an adverse effect on antibody activity. The interconversion of the acid peak and the main peak is manifested as a change in the acid peak and the main peak.

In this experiment, as shown in the data of table 6 above, from the IEC acid peak and main peak content data, the acid peak increase amplitude of the formulation No.10 sample after 2 weeks of storage was small, and the main peak content decrease amplitude was minimal, indicating that the formulation No.10 sample was stable after 2 weeks of storage. The stabilizer adopted by the No.10 preparation, namely trehalose, has good protection effect on the stability of the preparation sample under the illumination condition.

In addition, after 2 weeks of preservation, the main peak content of the preparation No. 7 sample is reduced to a smaller extent, and the acid peak growth is reduced to a smaller extent, which indicates that the preparation No. 7 sample has better stability after 2 weeks of preservation. Namely, arginine hydrochloride has better protection effect on the stability of the preparation sample. As can be seen from the data of formulation samples No. 8 and No. 9, glycine and methionine also have better protection for formulation samples.

Example 6 preparation of antibody A preparation

Based on the study described in examples 2-5 above, an antibody a formulation of the present invention was prepared using 0.2mg/ml polysorbate 80 as surfactant, his buffer as buffer, trehalose as stabilizer, and formulation pH of 5.5.

The antibody A preparation comprises trehalose, polysorbate 80, histidine hydrochloride, antibody A and water for injection.

The pharmaceutical preparation of the invention is prepared by the following method:

(1) The ingredients were weighed out of 850g trehalose dihydrate, 2g polysorbate 80, 2.8g histidine, 17.2g histidine hydrochloride. And 10L of water for injection was prepared for use.

(2) The weighed ingredients were dissolved in about 9L of water for injection (the order of addition of trehalose dihydrate, polysorbate 80, histidine hydrochloride may be selected at will), to prepare a solvent system.

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

(4) The initial formulation prepared as above was sterilized by passing through a 0.22 μm pore size hydrophilic polyvinylidene fluoride or polyethersulfone filtration membrane which can filter the formulation into a sterile container.

(5) After sterilization, the formulation is packaged for use, for example, in a prefilled syringe.

The volume of the formulation prepared as above and the weight of the materials employed may be scaled up or down, for example, 8L, 7L, 6L, 5L formulations comprising 80%, 70%, 60%, 50% of the recited weights, respectively.

Example 7 stability study of the antibody A preparation of the invention at high temperature (40 ℃ C.)

The stability of the antibody a formulation prepared in example 6 at high temperature was further examined. The antibody A preparation prepared as above was stored at a high temperature (40 ℃) for 15 days, and then subjected to capillary electrophoresis (CAPILLARY ELECTROPHORESIS, CE) detection using a capillary electrophoresis apparatus (Agilent, CE 7100) using a method as disclosed in Chen G et al ,Characterization ofglycoprotein biopharmaceutical products by Caliper LC90CE-SDS gel technology.Methods Mol Biol,2013,988(988):199-209, the results of the measurement are shown in FIG. 2.

As shown in fig. 2, the antibody a preparation was stored at high temperature (40 ℃) for 15 days, the main peak of the antibody CE decreased slowly from 98.11% to 96.79%, and 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 excellent protective effect on the antibody a therein.

EXAMPLE 8 Freeze-thaw stability study of antibody A formulations of the invention

Antibody A preparations of 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, by the method described in example 6 above. Each preparation was stored in a-80 ℃ refrigerator, then taken out and put in a constant temperature condition of 25 ℃ to be dissolved, and freeze thawing was repeated 5 times in this way, and the stability of the sample after 5 times of freeze thawing was examined. After 5 times freeze thawing, each preparation was diluted 1X 10 ⁵、1×10⁶ and 1X 10 ⁶ times, respectively, and ELISA was performed using a multifunctional microplate reader (Molecular Devices, spectramax M4) using the method disclosed in "isolation, purification and identification of anti-TNF-. Alpha.human single chain antibodies" as Wang Yuxiao, J.Immunol.2008 (6): 700-702. The 5 wells were loaded with antibody at each dilution and the recovery and Coefficient of Variation (CV) measured are set forth in Table 7.

TABLE 7 results of freeze thawing experiments on antibody A preparations with different antibody concentrations

In addition, SEC measurements were performed on each dilution of the formulations with different antibody concentrations using a high performance liquid chromatograph (Agilent, 1260 info) using the method disclosed by Zhao J et al (supra), see fig. 3.

The results show that after 5 times of freeze thawing, the concentration recovery rate of the antibody preparation with the concentration of the antibody A with the three levels of high, medium and low is 101.4% -107.3%, the variation coefficient is 6.1% -11.9%, and the SEC purity is above 98%. The antibody preparation with different antibody concentrations has stable quality after repeated freeze thawing for 5 times, and the preparation has good protection effect on the antibody A.

EXAMPLE 9 microbiological study

Further microbiological studies of the antibody a formulations of the invention were performed. Antibody a formulations were prepared as described above in example 6, and subjected to microbiological studies to determine if the formulations could 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 in an amount of 100cfu/ml, and after 14 days of storage at room temperature of 20-25 ℃, the total microbial growth of the preparations inoculated with each microorganism was examined in the inoculated containers. The index of evaluation was mainly 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 turbidity was unchanged indicating no microorganism growth. The data shown in Table 8 below indicate that after 14 days of storage at room temperature of 20-25 ℃, no microorganisms were observed under the microscope and that the turbidity of the formulation did not change, so that the formulation of the present invention did not support microbial growth.

TABLE 8 microbial detection results of antibody A preparations

Test index	Microorganism number cfu/ml	Turbidity/NTU
			Escherichia coli	0	Turbidity is unchanged
Staphylococcus aureus	0	Turbidity is unchanged
			Bacillus cereus	0	Turbidity is unchanged
Pseudomonas aeruginosa	0	Turbidity is unchanged

Example 10 pharmacodynamic comparative study of antibody A formulation and euphorbia

Pharmacodynamic studies of in vitro and in vivo animal models were performed on the antibody a formulation prepared as above. In which in vitro assays are mainly carried out in pharmacodynamic assays for binding to TNF-alpha, affinity to TNF-alpha, inhibition of the biological activity of TNF-alpha.

The affinity of the antibody A of the present invention for TNF- α was verified. The specific experimental procedure was as follows, the antibody A preparation prepared in example 6 above was diluted to 2. Mu.g/ml with PBS first, TNF-. Alpha.was diluted to 20nM with PBS (10602-HNAE, a science and technology Co., gmbH, beijing) and 2-fold gradient dilution was performed to give 20, 10, 5, 2.5, 1.25, 0.625, 0.3125nM dilutions of TNF-. Alpha.. The assay was performed using a BIAcore (GE HEALTHCARE, T200) using a ProteinA chip (GE HEALTHCARE, 29127556) and 2. Mu.g/ml of antibody A preparation was passed through the experimental flow path (Fc 2, fc 4) at a flow rate of 10. Mu.l/min, captured for 10s to a capture of about 200RU, after which the flow rate was adjusted to 30. Mu.l/min, TNF-. Alpha.dilutions of different concentrations were passed sequentially over the surfaces of the experimental flow path (Fc 2, fc 4) and the reference flow path (Fc 1, fc 3), respectively, with a binding time of 120s, a dissociation time of 600s, and finally a Glycine 1.5 (Shanghai Albumin Biotechnology Co., ltd., A110752) 60s for regeneration of the chip and into the next cycle. Analyzing the test result by using data analysis software Evaluation software3.1, carrying out double deduction on a reference flow path and a sample blank by using a sensing signal acquired by a sample test flow path, and carrying out dynamic fitting by using a 1:1 model to obtain dynamic parameters (ka: binding rate; kD: dissociation rate; kD: binding dissociation equilibrium constant) of each batch of samples and TNF-alpha. The same experiment was performed with commercially available euphorbia. The results show that the affinity constant of the antibody A preparation prepared in the invention for binding to TNF-alpha is about 60pM (the affinity constant of the antibody A preparation is 56.6 pM-67.4 pM, and the affinity constant of the antibody A preparation is 50.9 pM-72.1 pM), and the anti-TNF-alpha antibody preparation has strong affinity to TNF-alpha.

The affinity was measured using an antibody A standard (hereinafter referred to as standard S, standard S is 110.4mg/ml protein content, 1.980 X10 ⁵ U/mg active antibody A) and euphorbia using the same method as above, and the relative affinities of the antibody A of the present invention and a commercially available euphorbia antibody relative to standard S were further calculated. The results show that the relative affinities of the antibodies of the invention for binding to TNF- α are substantially identical for antibody a and euphoria, as shown in figure 4.

In addition, the binding capacity of the antibody A preparation prepared by the invention to TNF-alpha was verified (see Wang Yuxiao et al for test methods, isolation, purification and identification of anti-TNF-alpha human single chain antibodies J.Immunol.2008 (6): 700-702) and inhibition of TNF-alpha biological activity (see Trost L C et al .Acytotoxicity assay for tumornecrosis factor employing amultiwell fluorescence scanner.Analytical Biochemistry,1994,220(1):149-153). for test methods, which indicate that the EC50 for binding of the antibody A preparation prepared by the invention to TNF-alpha is about 8ng/ml and the EC50 for inhibition of TNF-alpha biological activity is about 6 ng/ml).

And further calculate the relative binding capacity and relative activity inhibition capacity of the antibody A preparation of the present invention with respect to the standard S and TNF-alpha. The relative binding capacity and relative activity inhibition capacity of the commercial euphorbia antibodies to the standard S and TNF-alpha were measured by the same method as above. The results are shown in fig. 5 and 6. As shown in FIGS. 5 and 6, the binding capacity of the antibody A preparation of the present invention and euphoria to TNF-alpha, and the inhibition of the biological activity of TNF-alpha by the antibody A preparation of the present invention and euphoria are substantially consistent.

The in vivo pharmacodynamic evaluation of antibody A prepared in accordance with the present invention was performed in a mouse model of human TNF transgenic idiopathic arthritis (Tg 197, biomedcode Hellas SA). The adopted injection mode is intraperitoneal injection, and the transgenic mice are divided into 5 groups, and 8 mice in each group are respectively injected with 1mg/kg euphoria (G1), physiological saline (G2), 1mg/kg antibody A (G3), 5mg/kg antibody A (G4) and 5mg/kg euphoria (G5). Injection was started from the third week, twice weekly, until the tenth week. A control group of transgenic mice (4) was added, and the group was sacrificed prior to the first dose, indicating an initial histopathological state. All experimental animals were sacrificed at week ten and the ankle was harvested for pathology assessment. The scoring system used for the in vivo arthritic lesion level and the pathology scoring system used for the ankle histopathology assessment are shown below.

In vivo arthritic lesion degree scoring system:

ankle joint histopathology assessment system:

the experimental results are shown in fig. 7A and 7B, which show that the antibody a of the present invention can inhibit arthritis onset of mice in the dosage ranges of 1mg/kg and 5mg/kg, and the antibody a of the present invention has therapeutic effects on tg197 arthritis model.

Example 11 comparison of molecular weight and glycosylation of antibody A of the present invention with commercially available euphorbia

The complete molecular weights and the deglycosylated molecular weights of antibody A and euphoria of the present invention were determined by the method recommended by the manufacturer using a high resolution mass spectrometer (Waters, xex G2-S QTof).

The antibody A of the present invention and the euphoria antibody were subjected to desugaring treatment. The antibody was diluted to about 1mg/ml with 50mM NH ₄HCO₃ (pH 8.0), N-glycosidase PNGaseF (Sigma Co., ltd., cat. No. P7367-50 UN) was added and reacted at 37℃for about 20 hours. Then, the molecular weight of the desugared antibody A and the molecular weight of the euphoria antibody are detected. The results are shown in fig. 8A and 8B.

The complete molecular weight comparison of the antibody A of the invention shown in FIG. 8A (i.e., the anti-TNF-. Alpha.antibody in the figure) with the euphorbia antibody shows that, unlike the antibody A of the invention, euphorbia is present at a molecular weight of 150178Da and several peaks at a larger number of molecular weights, which are lost after the desugaring treatment (FIG. 8B). It can be derived that the molecular weight difference between the antibody a of the present invention and the euphoric antibody is derived from the difference in the NGNA glycosylation level. The antibody a of the present invention has a lower NGNA glycosylation level than the euphorbia antibody, resulting in a difference in molecular weight between the two.

The average of three samples was determined by measuring the NGNA content (SpichtigV,Michaud J,Austin S.Determination of sialic acids in milks and milk-basedproducts[J].Analytical Biochemistry,2010,405(1):28-40.), in antibody a and euphorbia, and the results are shown in table 9, where the units are mol/mol of antibody, i.e. the molar amount of NGNA per mol of antibody.

TABLE 9 content of NGNA in antibody A and euphorbia

	Sample 1	Sample 2	Sample 3	Mean value of
					Antibody A	0.0043	0.0043	0.0039	0.0042
Euphorbia	0.29	0.35	0.29	0.31

For the amino acid sequences of the antibody A and the euphoric antibody, glycosylation modification mainly generates NGNA (N-glycolylneuraminic acid), and the NGNA glycosylation modification of TNF-alpha can cause the antibody to generate immunogenicity to human body after entering the human body, so as to generate adverse reaction. Compared with the euphoria antibody, the antibody A has lower glycosylation modification level of NGNA, so that the risk of adverse reaction of human body caused by NGNA is lower.

Example 12 comparison of Fc terminal Activity of antibody A of the invention with commercially available TNF-alpha euphoric antibody

Binding activity of the Fc-terminus of an antibody to a variety of cell surface receptors can affect a variety of properties of the antibody, such as binding of the Fc-terminus of an antibody to cell surface FcRn can affect the half-life of the antibody in humans, and binding of the Fc-terminus of an antibody to cell surface fcγriiia can affect ADCC (anti-DEPENDENT CELL-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity).

This experiment demonstrates the affinity of antibodies a and euphoria of the invention for FcRn. The specific protocol was followed by dilution of FcRn-biotin (ACRO Biosystems, cat#FCM-H82W 4) with PBS to 1. Mu.g/ml. SA Biosensor (Fertebio, cat#18-5019) was used to solidify the receptor protein in FcRn-biotin solution to a signal of about 1.2nm. The antibodies of the invention, antibody A and euphorbia were diluted to 200nM with PBS followed by a 2-fold gradient dilution to 3.125nM. PBS, drug dilutions, 1M MgCl ₂ (pH 8.3) solution, and PBST were sequentially added to the corresponding columns of 96-well plates, and FcRn-immobilized SABiosensor was used to detect baseline 60s in the Octet Platform form instrument (Fertebio, octet QKe), associate 60s in drug dilutions (200 nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125 nM) and sample blank (PBS), dissociate 60s in PBS, regenerate 5s in 1M MgCl ₂ (pH 8.3) solution, neutralize 5s in PBST. Wherein the regeneration, neutralization cycles were performed 3 times. Data were analyzed by treatment with DATAANALYSIS 8.2.2 (Fertebio), and sample data were fitted after subtraction of the sample blank (PBS) signal to give the affinity constant KD. The results show that the affinity constant of the antibody A prepared in the invention for binding to FcRn is about 7nM (antibody A is 6.35 nM-7.63 nM, and euphorbia is 6.09 nM-8.67 nM).

And the relative affinities of antibody a of the invention for FcRn binding to standard S were calculated. The above experiments were performed using euphorbia and the relative affinity of the euphorbia antibody to FcRn binding relative to standard S was calculated. The relative affinities of the antibodies of the invention for binding to FcRn are substantially identical for antibody a and euphorbia, the results are shown in figure 9.

In addition, the affinity of the antibodies of the invention a and euphoria with fcγriiia was also verified. Fcyriiia has been experimentally detected for both fcyriiia (F158) and fcyriiia (V158).

A specific experimental procedure for binding to FcgammaRIIIa (F158) was as follows, by dilution of FcgammaRIIIa (F158) -biotin (ACRO Biosystems, cat#CDA-H82E 8) to 1. Mu.g/ml. SABiosensor (Fertebio, cat#18-5019) was cured to a signal of about 0.6nm in fcγriiia (F158) -biotin dilution. The antibodies A and euphorbia of the invention were diluted to 500nM with PBS followed by a 2-fold gradient dilution to 7.812nM. PBS, drug dilutions, 1M MgCl ₂ (pH 8.3) solution, and PBST were sequentially added to the corresponding columns of 96-well plates, and the steps of baseline detection in PBS, association of 60s, dissociation of 60s in PBS, regeneration of 5s in 1M MgCl ₂ (pH 8.3) solution, and neutralization of 5s in PBST were performed in the Octet Platform instrument (Fertebio, octet QKe). The regeneration, neutralization cycles were performed 3 times. The data were analyzed by DATAANALYSIS 8.2.2 processing, and the sample data were fitted after subtraction of the sample blank (PBS) signal to give the affinity constant KD. The results show that the affinity constant of the binding of the antibody A prepared in the invention to FcgammaRIIIa (F158) is about 100nM (the antibody A is 110 nM-116 nM, and the euphoria is 93 nM-111 nM).

And the relative affinities of antibody a of the invention relative to standard S and fcγriiia (F158) were calculated. The above experiment was performed using euphorbia and the relative affinities of euphorbia antibodies to standard S and fcγriiia (F158) were calculated to be substantially identical for antibody a of the invention and euphorbia antibodies to fcγriiia (F158), the results are shown in fig. 10.

A specific experimental procedure for binding to FcgammaRIIIa (V158) was as follows, by dilution of FcgammaRIIIa (V158) -biotin (ACRO Biosystems, cat#CDA-H82E 9) to 1. Mu.g/ml. SABiosensor (Fertebio, cat#18-5019) was solidified in FcgammaR IIIa (V158) -Biotin dilutions to a signal of about 0.6nm. The antibodies A and euphorbia of the invention were diluted to 500nM with PBS followed by a 2-fold gradient dilution to 7.812nM. PBS, drug dilutions, 1M MgCl ₂ (pH 8.4), PBST were sequentially added to the corresponding columns of 96-well plates, and the steps of baseline detection in PBS, association in drug gradient dilutions (500 nM, 250nM, 125nM, 62.5nM, 31.25nM, 15.62nM, 7.812 nM) and sample blank (PBS), dissociation in PBS for 60s, regeneration in 1M MgCl ₂ (pH 8.4) for 5s, neutralization in PBST for 5s were performed. The regeneration, neutralization cycles were performed 3 times. The data were analyzed by DATAANALYSIS 8.2.2 processing, and the sample data were fitted after subtraction of the sample blank (PBS) signal to give the affinity constant KD. The results show that the affinity constant of the antibody A prepared in the invention for binding to FcgammaRIIIa (V158) is about 30nM (antibody A is 29.2 nM-40.8 nM, and euphorbia is 20.1 nM-42.2 nM).

And the relative affinities of antibody a of the invention relative to standard S and fcγriiia (V158) were calculated. The above experiment was performed using euphorbia and the relative affinities of the euphorbia antibody to the standard S and fcγriiia (V158) were calculated and the relative affinities of the euphorbia antibody a of the present invention and fcγriiia (V158) binding were substantially identical, the results are shown in fig. 11.

Example 13 pharmacokinetic comparison of antibody A of the invention with the commercially available anti-TNF-alpha antibody euphoria

The purpose of this example is to study the pharmacokinetics of antibody a of the invention and to provide a reference for future clinically reasonable dosing. The pharmacokinetic comparison of the antibody A of the present invention with the commercial anti-TNF-alpha antibody euphoria was also performed.

The study protocol was as follows:

6 cynomolgus monkeys (from Yongfu county, new Gui wild animal farming Co., ltd., 3-4.5 years old) were a low dose group of the antibody A of the invention, a high dose group of the antibody A of the invention, a low dose group of euphoria (Europe, from Janssen Biologics B.V.), a high dose group of euphoria (Europe), a low dose group of euphoria (America, from Janssen Biologics B.V.), and a high dose group of euphoria (America), each group of 6 animals, hermaphrodite halves, each low dose group being 3mg/kg, and each high dose group being 10mg/kg. The administration mode adopts subcutaneous injection, whole blood is collected 1h, 6h, 24h, 48h,3d, 4d, 5d, 7d and 9d after administration, plasma is separated, and the concentration of the drug in each sample is analyzed.

The method for measuring the antibody content in the plasma sample comprises the steps of firstly coating an ELISA plate with TNF-alpha protein (purchased from Beijing Yiqiao Shenzhou technology Co., ltd., product No. 10602-HNAE) capable of specifically binding to an anti-TNF-alpha antibody segment to capture the antibody in the biological sample, then adding an ELISA goat anti-human antibody (IgG-HRP, purchased from SIGMA, product No. A7164), incubating for about 1 hour at room temperature, allowing the ELISA goat anti-human antibody to bind to the captured anti-TNF-alpha antibody, finally adding a color development solution (purchased from BD bioscience Co., product No. 555214), standing for 15 minutes at room temperature to develop color, and reading the OD value of each well at 450nm by using Spectramax M4 (Molecular Devices) after termination of the reaction. The absorbance of the antibody A standard substance S and the concentration of the standard sample are fitted into a standard curve through four parameters, and the obtained standard curve is used for quantifying the antibody concentration of the sample to be detected, which is measured on the same ELISA plate. Pharmacokinetic parameters of the drug were calculated using the winnonlin6.4 non-compartmental model (NCA) and plasma drug mean concentration versus time curves were made and the results are shown in fig. 12.

The results show that at doses of 3mg/kg to 10mg/kg, the pharmacokinetic profile of the antibody A of the invention and euphorbia in cynomolgus monkeys is similar.

EXAMPLE 14 investigation of stability of antibody A formulations of the invention

The long-term stability of the antibody a formulation prepared as in example 6 at 5 ± 3 ℃ was further examined. The antibody A preparation prepared as above was stored at 5.+ -. 3 ℃ for 24 months under dark conditions, and the stability of antibody A was measured at 0 month, 3 months, 6 months, 9 months, 12 months, 18 months and 24 months, respectively, and the results are shown in Table 10.

As can be seen from the results in Table 10, the anti-TNF-alpha antibody has only small fluctuation of quality attributes such as SEC, IEC, CE and the like when being stored for 24 months under the condition of 5+/-3 ℃ and avoiding light, and has good stability, so that the anti-TNF-alpha antibody can be kept stable for at least 24 months in the anti-TNF-alpha antibody preparation of the invention.

Table 10 long term stability results at 5 ± 3 ℃ for antibody a formulation

Quality attributes	0 Month	3 Months of	6 Months of	9 Months of	12 Months of	18 Months of	24 Months of
								PH value of	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 Polymer 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-reducing 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 comprising an anti-TNF- α antibody comprising a heavy chain having the amino acid sequence shown in SEQ ID No.1 and a light chain having the amino acid sequence shown in SEQ ID No.2, and a pharmaceutically acceptable carrier comprising a stabilizer, a surfactant, a buffer, wherein the stabilizer is a sugar, arginine hydrochloride, glycine, methionine, or a combination thereof.

2. The antibody formulation of claim 1, having a pH of 4.5-6.5.

3. The antibody formulation of claim 1 or 2, wherein the concentration of the anti-TNF-a antibody is 5-130mg/ml.

4. The antibody formulation of any one of claims 1-3, wherein the concentration of the stabilizing agent is 160-265mM.

5. The antibody formulation of any one of claims 1-4, wherein the surfactant is polysorbate.

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

7. The antibody formulation of claim 1 or 2, wherein the buffer is one or more of histidine buffer, glutamic acid buffer, sodium acetate buffer, succinic acid buffer, and citric acid buffer.

8. The antibody formulation of any one of claims 1-7, wherein the buffer is at a concentration of 5-30mM.

9. An antibody formulation, the antibody formulation comprising:

5-130mg/ml of anti-TNF-alpha antibody,

160-265MM of a stabilizer, and,

5-30MM buffer, and

0.05-0.5Mg/ml surfactant,

The pH is 5.0-6.0.

10. An antibody formulation, the antibody formulation comprising:

100mg/ml of anti-TNF-alpha antibody,

225MM of trehalose, which is used as a reagent,

10MM histidine buffer, and

0.2Mg/ml polysorbate 80,

The pH is 5.0-5.8, preferably pH 5.5.

11. An antibody formulation, the antibody formulation comprising:

90-110mg/ml of anti-TNF-alpha antibody,

210-240MM of a stabilizing agent, and,

10-20MM buffer, and

0.1-0.3Mg/ml surfactant,

The pH is 4.5-6.0.

12. An antibody formulation, the antibody formulation comprising:

90-110mg/ml of anti-TNF-alpha antibody,

210-240MM of trehalose, and the concentration of the trehalose,

10-20MM histidine buffer, and

0.1-0.3Mg/ml polysorbate 80,

The pH is 4.5-6.0.

13. The antibody preparation of any one of claims 9-12, wherein the anti-TNF-a antibody comprises a heavy chain having the amino acid sequence shown in SEQ ID No.1 and a light chain having the amino acid sequence shown in SEQ ID No. 2.

14. A method for preparing the antibody preparation of any one of claims 1-13, comprising:

Weighing a stabilizer, a surfactant and a buffer;

dispersing the weighed components in a liquid solvent for injection to prepare a solvent system;

mixing the solvent system with the anti-TNF-alpha antibody.

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

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

17. A method of producing an anti-TNF- α antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID No.1 and a light chain comprising an amino acid sequence set forth in SEQ ID No.2, the method comprising

Culturing CHO cells containing a nucleic acid or vector encoding said anti-TNF- α antibody, such that said CHO cells express said anti-TNF- α antibody.

18. An anti-TNF- α antibody comprising a heavy chain comprising an amino acid sequence set forth in SEQ ID No.1 and a light chain comprising an amino acid sequence set forth in SEQ ID No.2, the molar ratio of Neu5Gc sialic acid (NGNA) levels of the anti-TNF- α antibody to the anti-TNF- α antibody being no more than 0.1.