CN110302377B

CN110302377B - Human antibody preparation for targeted therapy of TNF- α related diseases

Info

Publication number: CN110302377B
Application number: CN201910585391.0A
Authority: CN
Inventors: 吴用; 林键; 裴树军; 王盛武; 莫文俊; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2018-02-11
Filing date: 2019-02-03
Publication date: 2020-04-17
Anticipated expiration: 2039-02-03
Also published as: CN110302377A

Abstract

The invention relates to a human antibody preparation for targeted therapy of TNF- α related diseases, which is mainly a liquid water-containing pharmaceutical preparation, wherein the liquid water-containing pharmaceutical preparation can enhance the stability of TNF- α antagonist antibody medicines such as recombinant human anti-TNF- α monoclonal antibody and the like, and prolong the storage period of the medicines in the water-containing preparation.

Description

Human antibody preparation for targeted therapy of TNF- α related diseases

The patent application of the divisional application has the application date of 2 months and 3 days in 2019 and the application number of 201980000208.4, and is named as 'a human antibody preparation for targeted treatment of TNF- α related diseases'.

Technical Field

The invention relates to an antibody preparation for targeted therapy of TNF- α related diseases, which is mainly a liquid aqueous pharmaceutical preparation.

Background

Rheumatoid Arthritis (RA) is a common diseaseArthritis, the incidence of which in the population is 0.3% to 1%, can lead to bone destruction and joint dysfunction if not treated in time, and in the onset of RA, there are a number of proinflammatory cytokines involved, such as tumor necrosis factor- α (TNF- α), interleukins such as IL-1, IL-6, IL-8, etc^[1]. Therefore, inhibition of the production of proinflammatory cytokines or blocking their action is expected to improve the joint inflammatory response. In recent years, many new biological agents such as TNF inhibitors, anti-IL-1 antibodies, etc. have been developed to control disease progression by blocking or down-regulating the activity of inflammatory cytokines, and the main mechanisms of these biological agents are: (1) monoclonal antibodies directed against cytokines or their receptors; (2) soluble receptor antagonists, i.e., surface receptors for cellular factors that do not include transmembrane components and intracellular domains. The receptor antagonist binds to free cytokines, inhibiting the latter from binding to cell surface receptors. The half-life of soluble receptor antagonists is short and can be extended by the addition of certain integral structures such as Fc receptors for IgG or polyethylene glycol (PEG); (3) receptor antagonist proteins, biologically inactive proteins, compete with cytokines for binding to membrane receptors on cell surfaces. Receptor antagonist proteins must bind more than 90% of cell surface receptors to be effective.

At present, TNF- α (Tumor Necrosis Factor α, Tumor Necrosis Factor- α) is considered to be one of the most important proinflammatory cytokines among the cytokines of RA inflammatory response, and TNF- α plays an important role in the continuous development of RA lesions, local inflammatory response and tissue injury^[2]TNF- α and TNFR levels in RA are significantly elevated in serum, synovial membrane and synovial fluid, particularly in patients with severe disease and active phase, TNF- α levels in the serum of RA are positively correlated with joint injury score, Erythrocyte Sedimentation Rate (ESR) and anemia, TNF- α is also correlated with weight loss and disease recurrence in RA patients, TNF can act on various cells, such as macrophages can secrete inflammatory cytokines and chemokines, and inflammatory responses can be promoted, TNF- α has the main effects of (1) inducing endothelial cells to express adhesion molecules and Vascular Endothelial Growth Factors (VEGF), and promoting adhesion and infiltration of leukocytes and vascular endothelium, resulting in local inflammatory responses and vascular VEGF productionTNF- α acts on osteoclast, synoviocyte and chondrocyte to activate them, and produces metalloprotease, collagenase, stromelysin and prostaglandin E2(PGE2) to further destroy cartilage and cause bone erosion, joint inflammation and cartilage destruction, TNF- α can also promote the production of IL-1, IL-8 and TNF- α to aggravate tissue damage, so the inhibition of TNF- α is very important to control RA disease and improve prognosis^[3]。

TNF- α inhibitors currently approved by the U.S. FDA share 3 of the soluble receptor antagonist, Etanercept (Etanercept), the human murine chimeric antibody Infiniximab (Infliximab), and the humanized mAb Etanercept is a recombinant human type II tumor necrosis factor receptor-antibody fusion protein composed of a type II TNF receptor (p75) and the Fc portion of IgG1, Infliximab is a specific IgG1 monoclonal antibody to TNF (a chimera composed of a human Ig constant region and a murine Ig variable region), and the humanized mAb is also an IgG1 monoclonal antibody specific for TNF (both the Ig constant and variable regions are human)^[4-5]。

It is well known that pharmaceutical formulations must take into account stability, mode of administration, concentration, and the like. These factors affect the physicochemical degradation of the antibody during manufacture, storage and delivery, and thus risk to the applicator, and therefore a stable antibody formulation is necessary. In addition, certain antibody formulations require high dose administration and need to be formulated at higher concentrations, and high concentrations of antibody formulations may be associated with negative effects of antibody aggregation, reduced solubility, reduced stability, and the like. Currently, preparing high concentration antibody formulations still faces significant challenges.

Despite the need for such a human antibody BAT1406 formulation developed by the applicant for the treatment of TNF- α related diseases, with an antibody concentration of 20-80mg/mL (CN103446583A), it is difficult to exceed the upper limit of 80 mg/mL.

Disclosure of Invention

The present invention aims to provide a formulation that can support the presence of higher concentrations of antibody.

It is another object of the present invention to provide a more concentrated antibody preparation.

Another objective of the invention is to provide a more stable human antibody formulation for treating TNF- α related disorders.

The purpose of the invention is realized by the following technical means:

in one aspect, the invention provides an antibody preparation which is an aqueous pharmaceutical preparation comprising a recombinant human anti-TNF- α monoclonal antibody, a buffer system, a stabilizer, a surfactant.

(1) 20-120 mg/ml effective dose of anti-TNF-a humanized antibody

(2) Buffering agent

(3) 0.5-10% of stabilizer

(4)0.01 to 0.2 percent of surfactant

(5) Water for injection

The pH value of the antibody preparation is 4.0-7.0.

In one embodiment, the recombinant human anti-TNF- α monoclonal antibody is expressed in CHO cells by genetic engineering means and purified by a series of standard chromatographic steps after antibody preparation, followed by preparation of a pharmaceutical formulation, as a preferred embodiment, the recombinant human anti-TNF- α monoclonal antibody has a heavy chain as shown in SEQ ID No.1 and a light chain as shown in SEQ ID No.2 (accession number BAT1406) the TNF- α monoclonal antibody or an antigen-binding portion thereof, capable of binding to human TNF- α.

In a preferred embodiment, the concentration of the antibody is 40 to 120mg/ml, more preferably 50 to 100 mg/ml.

Wherein the buffer is selected from one or a combination of more of succinic acid, citric acid, His-HCl, acetic acid and phosphate; preferably, the buffer is selected from the group consisting of His-HCl, HAc, a combination of phosphate and HAc, a combination of His-HCl and HAc; more preferably a combination of His-HCl and HAc. In a preferred embodiment, each buffer is present in a concentration of 5 to 15 mM; more preferably 5 to 10 mM. More preferably, when the buffering agent is selected from the combination of two, the molar ratio of the former to the latter in the system is 1: 2-2: 1; more preferably 1: 1.

Wherein the stabilizer is 0.5 to 5.0 percent of Arg-HCl and 0.01 to 2 percent of Met; or is selected from 3.0 to 6.0 percent of sorbitol; or 7-10% of trehalose.

Wherein, the surfactant is selected from Tween80, Tween 20 or poloxamer 188.

The selected surfactants in the preparation belong to nonionic surfactants, and have the effect of improving the stability of the preparation, particularly under the conditions of oscillation and repeated freeze thawing. The inventors have found that different types of nonionic surfactants do not have much of an effect on stability enhancement in the formulations of the present invention. For example, Tween80, Tween 20 and poloxamer 188 belong to nonionic surfactants, the dosage of the Tween80, the Tween 20 and the poloxamer 188 in the biological monoclonal antibody preparation is 0.001% -0.1%, the dosage is very low, and the requirement on sample stability can be met by a very small amount of the surfactants. This may be because: the surfactant is an amphiphilic molecule with a hydrophilic group and a lipophilic group, the two phases are both regarded as the components of the phase through the affinity of different parts in the molecule with the two phases respectively, the molecules are arranged between the two phases, and the surfaces of the two phases are equivalently transferred into the interior of the molecule, so that the surface tension is reduced. As both phases are regarded as one component of the phase, the two phases are equivalent to that the two phases do not form an interface with the surfactant molecules, and the interface of the two phases is partially eliminated in this way, so that the surface tension and the surface free energy are reduced, and the aim of stabilizing the monoclonal antibody protein is fulfilled.

As a preferred embodiment, the antibody preparation of the invention comprises the following components:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5～10mM HAc

(3)5～10mM His-HCl

(4)2％～3.5％Arg-HCl

(5)0.01％～0.5％Met

(6)0.01％～0.1％Tween 80

(7) Water for injection

Wherein the pH of the preparation is 5.2-5.6;

or preferably, contains the following components:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5～10mM HAc

(3)5～10mM His-HCl

(4) 4.0-5% of sorbitol

(5)0.01％～0.1％Tween 80

(6) Water for injection

Wherein the pH of the preparation is 5.0-5.4;

or, preferably, the following components:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5～10mM HAc

(3)5～10mM His-HCl

(4)7 to 9 percent of trehalose

(5)0.01％～0.1％Tween 80

(6) Water for injection

Wherein the pH of the preparation is 5.0-5.4.

(1) 80-120 mg/ml of anti-TNF-alpha human antibody; preferably 85-120 mg/ml; more preferably

90～110mg/ml

(2)5～10mM HAc

(3)5～10mM His-HCl

(4)2％～3.5％Arg-HCl

(5)0.01％～0.5％Met

(6)0.01％～0.1％Tween 80

(7) Water for injection

Wherein the pH of the preparation is 5.2-5.6;

or preferably, contains the following components:

90～110mg/ml

(2)5～10mM HAc

(3)5～10mM His-HCl

(4) 4.0-5.0% of sorbitol

(5)0.01％～0.1％Tween 80

(6) Water for injection

Wherein the pH of the preparation is 5.0-5.4;

or, preferably, the following components:

(1) an effective amount of 80-120 mg/ml of anti-TNF-alpha humanized antibody; preferably 85-120 mg/ml; more preferably

90～110mg/ml

(2)5～10mM HAc

(3)5～10mM His-HCl

(4)7 to 9 percent of trehalose

(5)0.01％～0.1％Tween 80

(6) Water for injection

The pH value of the preparation is 5.0-5.4.

Alternatively, as a more preferred embodiment, the formulation comprises the following components:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5mM HAc

(3)5mM His-HCl

(4)3.1％Arg-HCl

(5)0.1％Met

(6)0.1％Tween 80

(7) Water for injection

Wherein the pH of the formulation is 5.4 + -0.2;

or more preferably, comprises the following components:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5mM HAc

(3)5mM His-HCl

(4) 4.2% sorbitol

(5)0.1％Tween 80

(6) Water for injection

Wherein the pH of the formulation is 5.2 + -0.2;

still or more preferably, the following components are contained:

(1) 45-120 mg/ml effective amount of anti-TNF-alpha humanized antibody

(2)5mM HAc

(3)5mM His-HCl

(4) 8.4% trehalose

(5)0.1％Tween 80

(6) Water for injection

The pH of the preparation is 5.2 +/-0.2.

In a preferred embodiment of the invention, the formulation comprises the following components:

(1) anti-TNF-alpha human antibody of 100mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4)3.1％Arg-HCl

(5)0.1％Met

(6)0.1％Tween 80

(7) Water for injection

Wherein the pH of the formulation is 5.4 + -0.2;

in another preferred embodiment of the invention, the formulation comprises the following components:

(1) anti-TNF-alpha human antibody of 50mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4)3.1％Arg-HCl

(5)0.1％Met

(6)0.1％Tween 80

(7) Water for injection

Wherein the pH of the formulation is 5.4 + -0.2;

(1) anti-TNF-alpha human antibody of 100mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4) 4.2% sorbitol

(5)0.1％Tween 80

(6) Water for injection

Wherein the pH of the formulation is 5.2 + -0.2;

(1) anti-TNF-alpha human antibody of 50mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4) 4.2% sorbitol

(5)0.1％Tween 80

(6) Water for injection

Wherein the pH of the formulation is 5.2 + -0.2;

(1) anti-TNF-alpha human antibody of 100mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4) 8.4% trehalose

(5)0.1％Tween 80

(6) Water for injection

The pH of the preparation is 5.2 +/-0.2.

(1) anti-TNF-alpha human antibody of 50mg/ml

(2)5mM HAc

(3)5mM His-HCl

(4) 8.4% trehalose

(5)0.1％Tween 80

(6) Water for injection

The pH of the preparation is 5.2 +/-0.2.

The formulations of the invention also contain NaOH for pH adjustment.

The osmotic pressure of the preparation is 150-400 mOsm/Kg; in a preferred embodiment, the osmotic pressure is 260 to 340 mOsm/Kg.

In another aspect, the present invention also provides a method for preparing the above antibody preparation, comprising the steps of:

⑴ preparing a formulation buffer containing a stabilizer;

⑵ the UF/DF ultrafiltration method is to use Millipore ultrafiltration membrane (regenerated cellulose, 30kDa molecular weight cut-off) to change the ultrafiltration liquid of the raw material drug (such as antibody BAT1406), change the preparation buffer solution with at least 8 volumes (namely the immediate volume of the sample of the changed liquid), detect the pH and conductance of the permeation end of the ultrafiltration system, and when the pH and conductance of the permeation end are consistent with those of the preparation buffer solution prepared just before, the change is sufficient, and the next step can be carried out, concentration.

⑶ after completion of the UF/DF step, a specific amount of surfactant is added to achieve the desired surfactant concentration.

⑷ the formulations were sterile filtered in an ultra clean bench using a 0.2 micron filter before filling the primary container.

The aqueous liquid antibody formulations of the present invention are suitable for subcutaneous injection.

In the antibody preparation, the concentration of the recombinant human anti-TNF- α monoclonal antibody BAT1406 can be as high as 120mg/ml, for example, the antibody can stably exist in the concentration range of 20-120 mg/ml, the highest concentration of the antibody in the BAT1406 antibody preparation in CN103446583A can only reach 80mg/ml (CN103446583A), and the upper limit of the antibody preparation is improved by 50%.

Rarely, the antibody preparation of the present invention also has high stability at high antibody concentrations, and is capable of extending the storage period of BAT1406 in an aqueous preparation; in particular, the formulation remains stable for at least 1 month at room temperature; can be stable for at least 36 months at 2-8 ℃; stability is maintained after at least 5 cycles of freeze-thawing. More specifically, BAT1406 was stable in aqueous formulations at room temperature for 1 month; 36 months at 2-8 ℃; after 5 cycles of freeze-thawing, EC50 is below 22ng/ml, more preferably below 21 ng/ml.

The recombinant human anti-TNF- α monoclonal antibody preparation developed by the invention has more stable high-concentration antibody protein and better tolerance to aggregation reaction caused by high-temperature change, so that the antibody preparation can be used for storing human antibodies related to targeted therapy of TNF- α, enhancing the stability of TNF- α antagonist antibody medicines such as recombinant human anti-TNF- α monoclonal antibody and the like, prolonging the storage period of the medicines in an aqueous preparation, and has important significance for targeted therapy of TNF- α related diseases.

Drawings

FIG. 1a buffer system screening SEC Main Peak analysis

FIG. 1b buffer system screening SEC fragment (mer) analysis

FIG. 2a analysis of IEC main peak screening by buffer system

FIG. 2b buffer system screening IEC acid peak analysis

FIG. 3a buffer screening CE (NR) Main Peak analysis

FIG. 3b buffer screening CE (NR) fragmentation (fragment) analysis

FIG. 4a high temperature (40 ℃) SEC Main Peak Change for the 100mg/ml formulation group

FIG. 4b high temperature (40 ℃) SEC Main Peak Change for 80mg/ml formulation group

FIG. 4c high temperature (40 ℃) SEC Main Peak Change for 50mg/ml formulation group

FIG. 5a high temperature (40 ℃) IEC main peak variation of 100mg/ml formulation group

FIG. 5b high temperature (40 ℃) IEC main peak variation of 80mg/ml formulation group

FIG. 5c high temperature (40 ℃) IEC main peak variation of 50mg/ml formulation group

FIG. 5d 100mg/ml formulation group high temperature (40 ℃) IEC acid peak changes

FIG. 5e 80mg/ml formulation group high temperature (40 ℃) IEC acid peak changes

FIG. 5f 50mg/ml formulation group high temperature (40 ℃ C.) IEC acid peak changes

FIG. 6a major peak Change in 100mg/ml formulation group light SEC

FIG. 6b major peak Change in illumination SEC for 80mg/ml formulation group

FIG. 6c major peak Change in illumination SEC for 50mg/ml formulation group

FIG. 7a variation of the major peaks of the 100mg/ml formulation group for IEC illumination

FIG. 7b variation of the major peaks of the 80mg/ml formulation group for IEC

FIG. 7c variation of the IEC main peak in the 50mg/ml formulation group by light

FIG. 7d 100mg/ml formulation group Lighting IEC acid peak Change

FIG. 7e 80mg/ml formulation group Lighting IEC acid peak Change

FIG. 7f 50mg/ml formulation group Lighting IEC acid peak Change

FIG. 8 number (. gtoreq.10 μm) of insoluble particles (pieces/mL) after dilution of different concentrations of surfactant samples

FIG. 9a Effect of different adjuvant formulations 40 ℃ high temperature on antibody (100mg/ml) (SEC main peak)

FIG. 9b Effect of different adjuvant formulations 40 ℃ high temperature on antibody (80mg/ml) (SEC main peak)

FIG. 9c Effect of different adjuvant formulations 40 ℃ high temperature on antibody (50mg/ml) (SEC main peak)

FIG. 10a Effect of different adjuvant formulations 40 ℃ high temperature on antibody (100mg/ml) (IEC Main Peak)

FIG. 10b Effect of different adjuvant formulations 40 ℃ high temperature on antibody (80mg/ml) (IEC Main Peak)

FIG. 10c Effect of different adjuvant formulations on antibody (50mg/ml) stability at 40 deg.C (IEC Main Peak)

FIG. 10d Effect of different adjuvant recipes high temperature at 40 ℃ on antibody (100mg/ml) (IEC acid peak)

FIG. 10e Effect of different adjuvant formulations 40 ℃ high temperature on antibody (80mg/ml) (IEC acid peak)

FIG. 10f Effect of different adjuvant formulations 40 ℃ high temperature on antibody (50mg/ml) stability (IEC acid)

FIG. 11a Effect of different adjuvant formulations 4000lx light on the stability of antibodies (100mg/ml) (SEC main peak)

FIG. 11b Effect of different adjuvant formulations 4000lx light on antibody (80mg/ml) stability (SEC main peak)

FIG. 11c Effect of different adjuvant formulations 4000lx light on antibody (50mg/ml) stability (SEC main peak)

FIG. 12a Effect of different adjuvant formulations 4000lx light on the stability of antibodies (100mg/ml) (IEC Main Peak)

FIG. 12b Effect of different adjuvant formulations 4000lx light on the stability of antibodies (80mg/ml) (IEC Main Peak)

FIG. 12c Effect of different adjuvant formulations 4000lx light on antibody (50mg/ml) stability (IEC Main Peak)

FIG. 12d Effect of different adjuvant formulations 4000lx light on antibody (100mg/ml) stability (IEC acid peak)

FIG. 12e Effect of different adjuvant formulations 4000lx light on antibody (80mg/ml) stability (IEC acid peak)

FIG. 12f Effect of different adjuvant formulations 4000lx light on antibody (50mg/ml) stability (IEC acid peak)

FIG. 13a antibody (100mg/ml) showing the change of SEC main peak at different pH values of three stabilizers (high temperature 40 ℃ C.)

FIG. 13b change of SEC Main Peak of antibody (80mg/ml) at different pH of three stabilizers (high temperature 40 ℃ C.)

FIG. 13c main peak change of SEC of antibody (50mg/ml) at different pH of three stabilizers (high temperature 40 ℃ C.)

FIG. 14a antibody (100mg/ml) IEC main peak variation at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 14b antibody (80mg/ml) IEC main peak variation at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 14c antibody (50mg/ml) IEC main peak variation at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 14d antibody (100mg/ml) IEC acid peak Change at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 14e antibody (80mg/ml) IEC acid peak change at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 14f IEC peak Change of antibody (50mg/ml) at different pH values of the three stabilizers (high temperature 40 ℃ C.)

FIG. 15a variation of the SEC main peak of the antibody (100mg/ml) at different pH values of the three stabilizers respectively (illumination 4000lx)

FIG. 15b variation of SEC Main Peak of antibody (80mg/ml) at different pH values of the three stabilizers respectively (illumination 4000lx)

FIG. 15c the SEC main peak variation of the antibody (50mg/ml) at different pH values of the three stabilizers respectively (illumination 4000lx)

FIG. 16a variation of the IEC main peak of the antibody (100mg/ml) at different pH values of the three stabilizers (illumination 4000lx)

FIG. 16b Change of IEC Main Peak of antibody (80mg/ml) at different pH values of the three stabilizers (illumination 4000lx)

FIG. 16c Change of IEC Main Peak of antibody (50mg/ml) at different pH values of the three stabilizers (illumination 4000lx)

FIG. 16d antibody (100mg/ml) IEC acid peak changes at different pH values for the three stabilizers (illumination 4000lx)

FIG. 16e Change of IEC acid peak of antibody (80mg/ml) at different pH values of the three stabilizers (illumination 4000lx)

FIG. 16f Change of IEC acid peak (illumination 4000lx) for antibody (50mg/ml) at different pH values of three stabilizers, respectively

FIG. 17a day 0 of Room temperature standing of formulation A1 in comparison to week 4 EC50

FIG. 17B day 0 of Room temperature standing of formulation B1 in comparison to week 4 EC50

FIG. 17C day 0 of Room temperature standing of formulation C1 in comparison to week 4 EC50

FIG. 17d preparation A2 standing at RT on day 0 in comparison with week 4 EC50

FIG. 17e formulation B2 standing at RT on day 0 vs. week 4 EC50

FIG. 17f preparation C2 standing at RT on day 0 vs. week 4 EC50

FIG. 17g formulation A3 standing at room temperature on day 0 vs. week 4 EC50

FIG. 17h formulation B3 standing at RT day 0 vs. week 4 EC50

FIG. 17i preparation C3 standing at RT on day 0 in comparison with week 4 EC50

FIG. 18a shows the in vivo metabolic processes of the recombinant human anti-TNF- α monoclonal antibody BAT1406, A1, B1 and C1 by subcutaneous injection

FIG. 18B subcutaneous injection of three formulations of A2, B2 and C2 of recombinant human anti-TNF- α monoclonal antibody BAT1406 for in vivo metabolism

FIG. 18C subcutaneous injection of three formulations of A3, B3 and C3 of recombinant human anti-TNF- α monoclonal antibody BAT1406 for in vivo metabolism

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

In the present invention, "%" referring to a component means a mass volume (w/v) percentage, for example, 6% of a certain component, that is, 6g of the component in 100ml of a formulation system, unless otherwise specified.

By "stable" herein is meant that the antibody (including antibody fragments thereof) does not aggregate, degrade, or fragment under a given production, preparation, transport, and/or storage condition, or only minimally, in a liquid formulation comprising the antibody (including antibody fragments thereof). A "stable" formulation retains biological activity under given manufacturing, preparation, shipping and/or storage conditions. The stability of the antibodies, including antibody fragments thereof, can be assessed by measuring the extent of aggregation, degradation, or fragmentation of the formulation, etc., by techniques such as SEC-HPLC, IEC-HPLC, CE-SDS, etc.

The buffer solution is prepared by a buffering agent, and can play a role in keeping the pH value of the solution relatively stable to a certain extent. Common buffers are, for example, salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; tris, tromethamine hydrochloride, or phosphate; amino acid component buffers such as glycine, histidine, and the like. Common buffer systems are for example: phthalate-hydrochloride buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, phosphate buffer (disodium hydrogen phosphate-sodium dihydrogen phosphate buffer), barbiturate sodium-hydrochloric acid buffer, Tris-HCl buffer, boric acid-borax buffer, glycine-sodium hydroxide buffer, glycine-hydrochloric acid buffer, borax-sodium hydroxide buffer, sodium carbonate-sodium bicarbonate buffer, and the like.

In theory, the above-mentioned buffer system can be applied to the antibody preparation of the present invention to maintain the pH value of the system stable. However, in practice, the choice of buffer will still critically affect the stability of the antibody formulations of the invention. Especially in the presence of high concentrations of antibodies, the choice of buffer system is more important.

The invention screens out a series of more suitable buffer solution systems through experiments, which are as follows: succinic acid buffer solution, citric acid buffer solution, histidine buffer solution, acetic acid buffer solution, phosphate buffer solution + citric acid buffer solution, phosphate buffer solution + acetic acid buffer solution, histidine buffer solution + succinic acid buffer solution, histidine buffer solution + citric acid buffer solution, histidine buffer solution + acetic acid buffer solution, succinic acid buffer solution + citric acid buffer solution, succinic acid buffer solution + acetic acid buffer solution. And the most preferable buffer liquid is histidine buffer solution + acetic acid buffer solution, experiments show that the SEC and IEC stability of the sample can be improved by the histidine component in the buffer solution, and the stable pH buffer range can be provided for the buffer system by the acetic acid component on the basis of not influencing the stability of the sample, so the histidine buffer solution + acetic acid buffer solution is preferable.

Histidine is a basic amino acid, and when preparing a histidine buffer, as a non-limiting example, the histidine buffer can be formed by dissolving the histidine in water and adjusting the pH with hydrochloric acid; wherein the added hydrochloric acid reacts with a portion of the histidine to form histidine hydrochloride, whereby histidine and histidine hydrochloride are present in the buffer. Histidine buffers with different pH values affect the different ratio of histidine to histidine hydrochloride, and the molar concentration of histidine is the sum of histidine and histidine hydrochloride. Therefore, in the present invention, histidine buffer is also denoted by the abbreviation "His-HCl".

The stabilizer in the present formulation has an effect of regulating osmotic pressure, and thus the stabilizer may also be referred to as an osmotic pressure regulator.

The preparation is a liquid aqueous medicine with the pH value of 4.0-7.0, contains 20-120 mg/ml of antibody, and has the effect of enhancing the stability of TNF- α antagonist antibody medicines containing recombinant human anti-TNF- α monoclonal antibody and the like, the concentration of the antibody of the preparation is lower than 20mg/ml, but the volume of the preparation needs to be increased according to the dosage, the dosage volume and the dosage frequency need to be reduced, the pain feeling of a patient can be reduced, and the infection risk caused by more dosage frequency can be controlled, so the concentration is preferably increased.

In some embodiments, the concentration of the antibody in the liquid aqueous pharmaceutical formulation is about 40-120 mg/ml, preferably 45-110 mg/ml, the antibody is recombinant human anti-TNF- α monoclonal antibody BAT1406, and BAT1406 is adalimumab.

The invention provides aqueous antibody preparations of different buffer systems, which comprise succinic acid buffer solution, citric acid buffer solution, histidine buffer solution, acetic acid buffer solution, phosphate buffer solution + citric acid buffer solution, phosphate buffer solution + acetic acid buffer solution, histidine buffer solution + succinic acid buffer solution, histidine buffer solution + citric acid buffer solution, histidine buffer solution + acetic acid buffer solution, succinic acid buffer solution + citric acid buffer solution, succinic acid buffer solution + acetic acid buffer solution, and the pH value is about 3.0-6.0.

The present invention provides liquid aqueous antibody formulations comprising a stabilizer (polyol) to regulate the osmotic pressure of the liquid system, stabilizing the antibody. The stabilizer is added to the antibody in an amount that may vary depending on the desired isotonicity of the formulation. In a preferred embodiment of the formulation of the invention, arginine hydrochloride and methionine are used as osmo-regulators in the formulation, and in a preferred embodiment of the invention, the stabilizer is selected from 3.0% to 6.0% sorbitol, preferably sorbitol at a concentration of 4.2%; or selected from 3.5% -5.5% mannitol, preferably mannitol concentration of 4.5%; or 0.9% sodium chloride; or 1.0 to 5.0 percent of proline, preferably the concentration of the proline is 2.5 percent; or 3.5 to 9 percent of cane sugar, preferably the concentration of the cane sugar is 6 percent; or 7.0 to 10 percent of trehalose, and the preferred concentration of the trehalose is 8.4 percent; or 0.5 to 5.0 percent of arginine hydrochloride, preferably the concentration of the arginine hydrochloride is 3.1 percent; or 0.1% -2% methionine. The osmotic pressure range of the preparation is 150-400 mOsm/Kg.

The present invention provides liquid aqueous antibody formulations comprising a buffer system. The buffer system comprises histidine buffer, acetic acid buffer or phosphate buffer. In a preferred embodiment of the formulation of the present invention, histidine buffer and acetic acid buffer are used as the buffer system, and the concentration of the histidine buffer and the concentration of the acetic acid buffer are both 5 to 15mM, preferably 5 to 10mM, and more preferably 5 mM. In another preferred embodiment of the formulation of the present invention, a histidine buffer, an acetate buffer and a phosphate buffer are used as the buffer system. One buffer in the scheme is histidine buffer, the concentration range is 5-15 mM, and the preferable concentration is 5 mM. The other buffer solution in the scheme is an acetic acid buffer solution, the concentration range is 10-25 mM, and the preferred concentration is 17 mM. The other buffer solution in the scheme is a phosphate buffer solution, the concentration range is 5-15 mM, and the preferred concentration is 10 mM. In another preferred embodiment of the formulation of the present invention, histidine buffer and acetate buffer are used as the buffer system. One buffer in the scheme is histidine buffer, the concentration range is 15-25 mM, and the preferred concentration is 20 mM. The other buffer solution in the scheme is an acetic acid buffer solution, the concentration range is 10-25 mM, and the preferred concentration is 17 mM. In another preferred embodiment of the formulation of the present invention, an acetate buffer and a phosphate buffer are used as the buffer system. One buffer solution in the scheme is an acetic acid buffer solution, the concentration range is 10-25 mM, and the preferred concentration is 17 mM. The other buffer solution in the scheme is a phosphate buffer solution, the concentration range is 5-15 mM, and the preferred concentration is 10 mM.

The invention provides liquid aqueous antibody formulations comprising a surfactant. Typical surfactants include nonionic surfactants such as Tween 80. The surfactant reduces aggregation of the produced antibody and/or reduces particle formation in the formulation and/or reduces adsorption. In an alternative embodiment of the formulation of the invention, the formulation is surfactant Tween 80. In a preferred embodiment, the formulation includes about 0.01% to about 0.2% Tween80, more preferably about 0.01% to about 0.1%, and even more preferably about 0.1% Tween80 is present in the formulations of the invention.

In a preferred embodiment of the present invention, the present invention provides a liquid aqueous antibody formulation combination regimen wherein the formulation buffer system comprises a histidine buffer and an acetic acid buffer. The stabilizer of the formulation comprises sorbitol, or trehalose, or a combination of arginine hydrochloride and methionine. The surfactant of the formulation includes Tween 80. The pH value of the preparation ranges from 4.0 to 7.0. The osmotic pressure range of the preparation is 150-400 mOsm/Kg. In one embodiment of the present invention, histidine buffer and acetic acid are used as the buffer system, and the concentration ranges from 5mM to 15mM, preferably from 5mM to 10mM, more preferably from 5mM, and the pH range is from 4.0 to 7.0, preferably from 5.2 to 5.6. Arginine hydrochloride and methionine as stabilizers in the concentration ranges of 0.5-5.0% and 0.01-2%, respectively, preferably in the concentrations of 3.1% and 0.1%, respectively. Tween80 is used as a surfactant at a concentration ranging from 0.01% to 0.2%, preferably at a concentration of 0.1%. The osmotic pressure range of the preparation is 150-400 mOsm/Kg, and the optimized osmotic pressure is 260-340 mOsm/Kg. In another preferred embodiment of the formulation of the present invention, histidine buffer and acetic acid are used as the buffer system, and the concentration ranges from 5mM to 15mM, preferably from 5mM to 10mM, more preferably from 5mM, and the pH range is from 4.0 to 7.0, preferably from 5.0 to 5.4. Sorbitol as a stabilizer is present in a concentration range of 3% to 6%, preferably 4.2%. Tween80 is used as a surfactant at a concentration ranging from 0.01% to 0.5%, preferably at a concentration of 0.1%. The osmotic pressure range of the preparation is 150-400 mOsm/Kg, and the preferred osmotic pressure is 260-340 mOsm/Kg. In another preferred embodiment of the formulation of the present invention, histidine buffer and acetic acid are used as the buffer system, and the concentration ranges from 5mM to 15mM, preferably from 5mM to 10mM, more preferably from 5mM, and the pH range is from 4.0 to 7.0, preferably from 5.0 to 5.4. Trehalose is used as a stabilizer in a concentration range of 7% to 10%, preferably 8.4%. Tween80 is used as a surfactant at a concentration ranging from 0.01% to 0.2%, preferably at a concentration of 0.1%. The osmotic pressure of the preparation is 150-400 mOsm/Kg, and the preferable osmotic pressure is 260-340 mOsm/Kg.

The present invention provides liquid aqueous pharmaceutical formulations comprising an antibody suitable for therapeutic use, which pharmaceutical formulation is convenient to administer and contains a high protein concentration, primarily for the treatment of conditions caused by TNF- α. in one embodiment, the pharmaceutical formulation has the effect of enhancing stability. in another embodiment, the formulation of the invention is stable after at least 5 freeze-thaw cycles. in another embodiment, the formulation of the invention remains stable after being placed at room temperature for 1 month. in another embodiment, the formulation of the invention remains stable for at least 36 months at 2-8 deg.C.

In another embodiment of the invention, the liquid aqueous pharmaceutical formulation comprises an antibody or antigen-binding portion thereof which is a recombinant antibody or antigen-binding portion thereof which inhibits human TNF- α -induced expression of ELAM-1 on human venous endothelial cells in another embodiment, the formulation comprises recombinant human anti-TNF- α monoclonal antibody BAT1406 having 2 heavy chains as set forth in SEQ ID No.1 and 2 light chains as set forth in SEQ ID No. 2.

The invention provides an aqueous pharmaceutical formulation comprising an effective amount of an antibody component, the buffer system is a histidine buffer and an acetic acid buffer, the osmotic pressure stabilizer is arginine hydrochloride and methionine, or sorbitol, or trehalose, the surfactant Tween80 and water for injection, the pH is about 4.0-7.0. In one embodiment of the invention, the formulation is suitable for subcutaneous injection. In one embodiment of the invention, the concentration of said antibody in said liquid aqueous pharmaceutical formulation is about 100 mg/ml; in one embodiment of the invention, the concentration of said antibody in said liquid aqueous pharmaceutical formulation is about 80 mg/ml; in another embodiment of the invention, the concentration of said antibody in said liquid aqueous pharmaceutical formulation is about 50 mg/ml.

In a preferred embodiment of the invention, the formulation is a solution containing the ingredients shown in tables 1a-1c, preferably the formulation is contained in a pre-filled syringe, wherein the formulation may contain 40mg of an effective amount of anti-hTNF- α antibody, 3.1% arginine hydrochloride with 0.1% methionine, 0.1% Tween80, 5mM histidine hydrochloride and 5mM acetic acid, sodium hydroxide for pH adjustment and water for injection (see Table 1a), wherein the formulation may also contain 40mg of an effective amount of anti-hTNF- α antibody, 4.2% sorbitol, 0.1% Tween80, 5mM histidine hydrochloride and 5mM acetic acid, sodium hydroxide for pH adjustment and water for injection (see Table 1b), wherein the formulation may also contain 40mg of an effective amount of anti-hTNF- α antibody, 8.4% trehalose, 0.1% Tween80, 5mM histidine hydrochloride and 5mM acetic acid, sodium hydroxide for pH adjustment and water for injection (see Table 1 c).

TABLE 1a. list of A1 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 100mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Arginine hydrochloride	3.1％	Osmotic pressure regulator
			Methionine	0.1％	Osmotic pressure regulator
Histidine hydrochloride	5mM	Buffering agent
			Acetic acid	5mM	Buffering agent
Tween80	0.1％	Surface active agent
			Sodium hydroxide	Adjusting pH to 5.4 + -0.2	pH regulator
Water for injection	Make up to 400ul	Solvent(s)

TABLE 1B list of B1 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 100mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Sorbitol	4.2％	Osmotic pressure regulator
			Histidine hydrochloride	5mM	Buffering agent
Acetic acid	5mM	Buffering agent
			Tween80	0.1％	Surface active agent
Sodium hydroxide	Adjusting pH to 5.2 + -0.2	pH regulator
			Water for injection	Make up to 400ul	Solvent(s)

TABLE 1C list of C1 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 100mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Trehalose	8.4％	Osmotic pressure regulator
			Histidine hydrochloride	5mM	Buffering agent
Acetic acid	5mM	Buffering agent
			Tween80	0.1％	Surface active agent
Sodium hydroxide	Adjusting pH to 5.2 + -0.2	pH regulator
			Water for injection	Make up to 400ul	Solvent(s)

In another preferred embodiment of the invention, the formulation comprises a solution of the ingredients shown in tables 1d-1 f; preferably, the formulation is contained in a pre-filled syringe.

TABLE 1d list of A2 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 80mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Arginine hydrochloride	3.1％	Osmotic pressure regulator
			Methionine	0.1％	Osmotic pressure regulator
Histidine hydrochloride	5mM	Buffering agent
			Acetic acid	5mM	Buffering agent
Tween80	0.1％	Surface active agent
			Sodium hydroxide	Adjusting pH to 5.4 + -0.2	pH regulator
Water for injection	Make up to 500ul	Solvent(s)

TABLE 1e list of B2 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 80mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Sorbitol	4.2％	Osmotic pressure regulator
			Histidine hydrochloride	5mM	Buffering agent
Acetic acid	5mM	Buffering agent
			Tween80	0.1％	Surface active agent
Sodium hydroxide	Adjusting pH to 5.2 + -0.2	pH regulator
			Water for injection	Make up to 500ul	Solvent(s)

TABLE 1f list of C2 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 80mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Trehalose	8.4％	Osmotic pressure regulator
			Histidine hydrochloride	5mM	Buffering agent
Acetic acid	5mM	Buffering agent
			Tween80	0.1％	Surface active agent
Sodium hydroxide	Adjusting pH to 5.2 + -0.2	pH regulator
			Water for injection	Make up to 500ul	Solvent(s)

In another preferred embodiment of the invention, the formulation comprises a solution of the ingredients shown in tables 1g-1 i; preferably, the formulation is contained in a pre-filled syringe.

TABLE 1g A3 formulation component List of the recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 50mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Arginine hydrochloride	3.1％	Osmotic pressure regulator
			Methionine	0.1％	Osmotic pressure regulator
Histidine hydrochloride	5mM	Buffering agent
			Acetic acid	5mM	Buffering agent
Tween80	0.1％	Surface active agent
			Sodium hydroxide	Adjusting pH to 5.4 + -0.2	pH regulator
Water for injection	Make up to 800ul	Solvent(s)

TABLE 1h list of B3 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 50mg/mL)

Name of ingredient	Dosage of	Function(s)
			Active substance: antibody BAT1406	40mg	Active substance
Sorbitol	4.2％	Osmotic pressure regulator
			Histidine hydrochloride	5mM	Buffering agent
Acetic acid	5mM	Buffering agent
			Tween80	0.1％	Surface active agent
Sodium hydroxide	Adjusting pH to 5.2 + -0.2	pH regulator
			Water for injection	Make up to 800ul	Solvent(s)

TABLE 1i list of C3 formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406 (antibody concentration 50mg/mL)

In summary, the present invention provides a liquid aqueous drug, as an embodiment, the formulation may comprise 40 to 120mg/ml antibody concentration, 5 to 15mM histidine buffer, 5 to 15mM acetic acid buffer, 0.5 to 5.0% arginine hydrochloride, 0.01 to 2% methionine, 0.01 to 0.2% Tween80 and water for injection, and the pH is 4.0 to 7.0. The preferable scheme comprises antibody concentration of 45-110 mg/ml, 5mM histidine buffer solution, 5mM acetic acid buffer solution, 3.1% arginine hydrochloride, 0.1% methionine, 0.1% Tween80 and water for injection, pH is 5.4 +/-0.2, sodium hydroxide for adjusting pH and water for injection (hereinafter referred to as A preparation).

In another embodiment, the formulation may contain 40-120 mg/ml antibody concentration, 5-15 mM histidine buffer, 5-15 mM acetic acid buffer, 3.0-6.0% sorbitol, 0.01-0.2% Tween80 and water for injection, pH 4.0-7.0. The preferable scheme comprises antibody concentration of 45-110 mg/ml, 5mM histidine buffer solution, 5mM acetic acid buffer solution, 4.2% sorbitol, 0.1% Tween80 and water for injection, wherein the pH is 5.2 +/-0.2, and sodium hydroxide and water for injection (hereinafter referred to as the preparation B) for adjusting the pH.

In another embodiment, the formulation may contain 40-120 mg/ml antibody concentration, 5-15 mM histidine buffer, 5-15 mM acetic acid buffer, 7-10% trehalose, 0.01-0.2% Tween80 and water for injection, pH 4.0-7.0. The preferable scheme comprises antibody concentration of 45-110 mg/ml, 5mM histidine buffer solution, 5mM acetic acid buffer solution, 8.4% trehalose, 0.1% Tween80 and water for injection, pH is 5.2 +/-0.2, and sodium hydroxide and water for injection (hereinafter referred to as preparation C) for adjusting pH.

The preparation is stable after at least 5 times of freeze-thaw cycles, can be kept stable after being placed for 36 months at the temperature of 2-8 ℃, is mainly used for treating diseases caused by TNF- α, is suitable for subcutaneous injection, and can enhance the stability of TNF- α antagonist antibody medicines such as recombinant human anti-TNF- α monoclonal antibody and the like and prolong the storage period of the medicines in the aqueous preparation.

Note: histidine hydrochloride, histidine buffer, histidine, His-HCl are understood herein to mean the same, i.e. a buffer configured with histidine and hydrochloric acid.

Examples

Example 1: effect of buffer systems on formulation stability

The combination of 11 groups of buffer systems containing antibodies is shown in table 2.

The preparation method of the group 11 antibody-containing buffer system comprises the following steps:

the sample components were: BAT1406 antibody was added to 10mM buffer at an antibody concentration of 50 mg/mL. Since the experiment was performed with the screening buffer, no other components were added at all.

The preparation method comprises the following steps: 10mM of buffer is weighed, dissolved in water, adjusted to pH 5.2 with sodium hydroxide and added to volume. Finally, 11 groups of samples are prepared by adopting an ultrafiltration method. The ultrafiltration method is as described above.

In a certain pH range, the stability of the recombinant human anti-TNF- α monoclonal antibody BAT1406 is studied at 0, 5, 10 and 15 days respectively, and the SEC main peak, the aggregate and the fragment are analyzed respectively, the IEC main peak and the acid region are analyzed, the CE (NR) main peak and the fragment are analyzed, and DSC detection is carried out.

Analytical method by SEC-HPLC:

1, a liquid chromatography system and a TOSOH biotech TSK-GEL G3000SWXL chromatography column (column size 7.8 × 300mm, 5 μm) were prepared. Starting a liquid chromatograph, starting instrument operation software, calling a method, starting a pump to pump in a mobile phase, setting the wavelength of an ultraviolet detector, setting the column temperature to be room temperature, adjusting the flow rate to be 0.5ml/min, and balancing for 30-60min until the baseline is stable.

And 2, feeding a sample, recording a chromatogram, and calculating the percentage content of the monomer or polymer of the test solution according to a peak area normalization method after integration.

Analytical method for IEC-HPLC:

1, preparing a liquid chromatography system and a ProPac WCX-10 weak acid cation exchange separation column. And (3) starting a power supply, starting a liquid phase data analysis system, calling a method, starting a pump, balancing the chromatographic column under the initial conditions of 94% of mobile phase A and 0.2ml/min, and after the pressure is stable, adjusting the flow rate to 0.8ml/min for balancing for 30-60min until the baseline is stable.

And 2, injecting a sample, manually integrating the detected chromatogram, and calculating the total percentage content of the three lysine variants (lys0, lys1 and lys2) according to a peak area normalization method.

Analysis method of CE-SDS (NR):

the capillary electrophoresis apparatus adopts PA800 plus of Beckman, after sample introduction, a chromatogram is recorded, data are processed by integration, and the percentage content of a monomer peak is calculated by adopting an area normalization method.

Analytical method by DSC:

starting up the machine for preheating, cleaning the pipeline, diluting the sample, degassing and sampling.

And 2, opening an operation program on the computer desktop, setting parameters and detecting.

And 3, performing fitting analysis on the detected peak images.

TABLE 2 buffer System Components

Note: table 1 with numbers 1-11 respectively corresponds to numbers A160923-1-A160923-11 in the figure

The results show that the main peaks of prescriptions 3, 4, 6, 7 and 9 have less descending amplitude and better stability as can be seen from the graph plotting of SEC main peak data; 2. the main peak of prescriptions No. 5, 8 and 10 is greatly reduced, the stability is poor, and the result is shown in figure 1 a; as can be seen from the SEC fragment data plots, there was less increase in SEC fragments for prescriptions No. 3, 4, 6, 9, 11 and more increase in SEC fragments for prescriptions No.2, 5, 8, 10, with the results shown in fig. 1b.

As can be seen from the IEC main peak data mapping, the IEC main peak of the prescriptions No. 3 and No. 4 has less reduction range and better stability; 1. the main peaks of the prescriptions No.2, 10 and 11 are reduced more, and the stability is poorer, and the result is shown in figure 2 a; as can be seen from the IEC acid region data plot, the acid regions of formulas 3 and 5 increased less, and the acid regions of

formulas

1, 10 and 11 increased more, and the results are shown in FIG. 2 b.

From the main peak data of ce (nr), the main peaks of samples No. 3, 4, 6, and 9 were reduced less, and the main peaks of samples No.1, 2, 5, 8, and 10 were reduced more, as shown in fig. 3 a; from the Fraction data of CE (NR), the Fraction growth was smaller for samples Nos. 3, 4, 6, and 9, and the Fraction increase was greater for samples Nos. 2, 5, 8, and 10, as shown in FIG. 3 b.

The results are shown in Table 3, in which the Tm values of the samples Nos. 4, 6, 9 and 11 are high and the Tm values of the samples Nos. 1, 2, 3, 8 and 10 are low, as seen from the Tm value data of DSC.

TABLE 3 Tm values of DSC samples

Sample name	Buffer system	Tm(℃)
			1	Succinic acid buffer solution	73.59
2	Citric acid buffer	72.68
			3	Histidine buffer	73
4	Acetic acid buffer solution	74.51
			5	Phosphate buffer + citric acid buffer	73.63
6	Phosphate buffer + acetic acid buffer	74.42
			7	Histidine buffer + succinic acid buffer	73.44
8	Histidine buffer + citric acid buffer	72.71
			9	Histidine bufferWashing solution and acetic acid buffer solution	73.84
10	Succinic acid buffer + citric acid buffer	72.99
			11	Succinic acid buffer solution + acetic acid buffer solution	73.94

Differential scanning calorimetry

In summary, from the analysis of the Tm values of the SEC main peak, fragment, IEC main peak, acid region, CE (NR) main peak, fragment and DSC, buffer systems No. 3, 4, 6 and 9 can be selected, and the specific situation is shown in Table 4.

TABLE 4 optional buffer system Components and pH ranges

Serial number	Buffer system	pH range
			3	Histidine buffer	5.5～6.5
4	Acetic acid buffer solution	3.6～5.8
			6	Phosphate buffer + acetic acid buffer	4.0～6.0
9	Histidine buffer + acetic acid buffer	4.0～6.0

Example 2: effect of different buffer combinations on antibody stability

BAT1406 antibody preparations were prepared according to the components in Table 5, placed at 40 ℃ for 4 weeks, and samples were analyzed for SEC-HPLC main peak, multimer, fragment, and IEC-HPLC main peak, acid region, base peak 2, and base peak 3, respectively.

TABLE 5 ingredient tables of the respective recipes

The preparation method of the preparation sample comprises the following steps:

a method of preparation comprising the steps of:

⑴ preparation of buffer solution, taking preparation of 1L respectively as an example, the combined preparation method of the buffer agents No.1 to No. 4 comprises the steps of weighing 1.05g of histidine hydrochloride and 0.834g of acetic acid, dissolving in water, adjusting the pH value to 5.2 by using sodium hydroxide, and fixing the volume to 1L, weighing 2.10g of histidine hydrochloride, 2.836g of acetic acid and 1.38g of disodium hydrogen phosphate, dissolving in water, adjusting the pH value to 5.2 by using sodium hydroxide, and fixing the volume to 1L, weighing 4.19g of histidine hydrochloride and 2.836g of acetic acid, dissolving in water, adjusting the pH value to 5.2 by using sodium hydroxide, fixing the volume to 1L, weighing 2.836g of acetic acid and 1.38g of disodium hydrogen phosphate, dissolving in water, adjusting the pH value to 5.2 by using sodium hydroxide, and fixing the volume to 1L.

⑵ it adopts UF/DF ultrafiltration method, uses ultrafiltration membrane (regenerated cellulose, 30kDa molecular weight cut-off) to change the ultrafiltration of the raw material drug (such as antibody BAT1406), changes the preparation buffer solution with at least 8 volumes (i.e. the immediate volume of the sample of the changed solution), detects the pH and conductance of the permeation end of the ultrafiltration system, when the pH and conductance of the permeation end are consistent with those of the preparation buffer solution prepared just now, it indicates that the solution change is sufficient, and can enter the next step, concentration.

(3) Prior to filling the primary container, the formulation was sterile filtered in a clean bench using a 0.2 micron filter. Filling is also performed aseptically.

As can be seen from the SEC main peak data, the main peaks of prescriptions No.1 and No. 4 decrease slowly, and the main peaks of prescriptions No.2 and No. 3 degrade faster; as can be seen from the IEC main peak data, the main peak of prescription No.1 is reduced slowly, and the main peaks of prescription Nos. 2, 3 and 4 are degraded slightly quickly; from the IEC acid region data, it can be seen that the acid region of formula 4 grows faster, and the acid region of

formula

1, 2, and 3 grow in the same direction. And combining the results, and finally selecting the prescription No.1, wherein the specific results are shown in Table 6.

TABLE 6a SEC-HPLC and IEC-HPLC analysis results for each prescription of the 100mg/ml formulation

TABLE 6b SEC-HPLC and IEC-HPLC analysis results for each formulation of 80mg/ml formulation

TABLE 6c SEC-HPLC and IEC-HPLC analysis results for each prescription of 50mg/ml formulation

The sample names A161224-1/A161224-2/A161224-3/A161224-4 in Table 6 correspond to the reference numbers 1/2/3/4 in Table 5, respectively.

Example 3: stabilizer screening study

The experiment with 11 samples and the results thereof are shown below.

All 11 samples contained: stabilizers (see table 7 below for details) + buffer (5mM acetic acid +5mM histidine buffer) + surfactant (0.1% tween 80) + BAT1406 antibody. The antibody concentrations were 100mg/mL (high concentration), 80mg/mL (medium concentration), and 50mg/mL (low concentration), respectively.

Stabilizer screening experiments (high temperature 40 ℃): the analysis of the SEC main peak, IEC main peak and acid region was carried out on 11 stabilizers (see Table 7) at

days

0, 5, 10 and 15 at a high temperature of 40 ℃. The detection method was the same as in example 1.

TABLE 7 stability Components

Serial number	Sample name	Stabilizer	Concentration of stabilizer
				1	A161209-1	--	--
2	A161209-2	Sorbitol	4.20％
				3	A161209-3	Mannitol	4.50％
4	A161209-4	Sodium chloride	0.90％
				5	A161209-5	Proline	2.50％
6	A161209-6	Sucrose	6％
				7	A161209-7	Trehalose	8.4％
8	A161209-8	Arginine hydrochloride	3.1％
				9	A161209-9	Glycine	1％
10	A161209-10	Methionine	0.1％
				11	A161209-11	Calcium chloride	10mM

For the analysis results of the concentrations of the high, medium and low antibodies, the change trends of the SEC main peaks of 11 groups of samples are consistent as can be seen from the graph plotting of the SEC main peak data, wherein the main peaks of prescriptions 1 and 4 have larger descending amplitude, the main peak degradation trends of other prescriptions have small difference, and the results are shown in FIGS. 4 a-c; as can be seen from the IEC main peak data mapping, the change trends of the IEC main peaks of 11 groups of samples are consistent, wherein the main peaks of prescriptions No. 4 and No. 8 have smaller descending amplitude, and the main peak changes of other prescriptions have little difference, and the results are shown in FIGS. 5 a-c; as can be seen from the IEC acid region data plot, the change trend of the IEC acid regions of the 11 groups of samples was consistent, the increase of the acid regions of the formulas No. 4 and No. 8 was small, and the change of the acid regions of the other formulas was not very different, and the results are shown in FIGS. 5 d-f.

Stabilizer screening experiment (illumination 4000 lx): the samples containing different stabilizers (see Table 7) were analyzed at

day

0, 5, 10, 15 under 4000lx illumination for the main SEC peak, the main IEC peak and the acid region, CE-SDS (NR). The detection method is shown in the examples.

The analysis results of the concentrations of the high, medium and low antibodies show that the main peak of the stabilizer No. 10 has the minimum change, the main peak of the formula No. 4 has the maximum descending amplitude, the descending trends of the main peaks of other groups of stabilizers have little difference from each other by plotting SEC main peak data, and the results are shown in FIGS. 6 a-c; as can be seen from the IEC main peak data plot, the main peak of the stabilizer No. 10 has the smallest descending amplitude, the main peaks of the stabilizers No. 4 and 5 have larger descending amplitude, and the descending trends of the main peaks of the stabilizers in other groups are consistent, and the results are shown in FIGS. 7 a-c; as can be seen from the IEC acid region data plot, the acid region growth of the stabilizer No. 10 is the smallest, the acid region growth of the stabilizers No. 5 and 9 is larger, and the acid region growth trends of the stabilizers in other groups are consistent and the results are shown in FIGS. 7 d-f; as can be seen from the main peak data of CE-SDS (NR), the main peak of prescription No. 10 was reduced the least, and the main peak of the other prescriptions were reduced consistently, and the specific results are shown in Table 8.

TABLE 8a stabilizer screening CE-SDS (NR) assay results (antibody concentration: 100mg/ml)

TABLE 8b stabilizer screening CE-SDS (NR) assay results (antibody concentration: 80mg/ml)

TABLE 8c stabilizer screening CE-SDS (NR) assay results (antibody concentration: 50mg/ml)

According to the analysis results of SEC, IEC, CE-SDS (NR) at 40 ℃ and 4000lx of illumination, the concentration of the high, medium and low antibodies has consistent trend, the No. 4 (sodium chloride), the No. 5 (proline) and the No. 9 (glycine) are unstable at high temperature and under illumination, and other groups are stable and prominent, and the No. 8 (arginine hydrochloride), the No. 10 (methionine) stabilizer, the No.2 (sorbitol) and the No. 7 (trehalose) can be preferably selected.

Example 4: surfactant screening

This experiment was performed to compare the protective effect of 3 surfactants on antibody proteins to determine how much surfactant concentration is effective in protecting antibody stability.

Protein solutions containing different concentrations of surfactant were prepared. The specific formulation is shown in Table 9. The antibody protein concentration in the solution was 100mg/mL, 80mg/mL, 50mg/mL, the protein (BAT1406) contained no surfactant, and the other components (5mM HAc, 5mM HIS-HCl, 3.1% Arg-HCl, 0.1% Met, pH5.3) were contained.

TABLE 9 protein solutions of different formulations

Serial number	Prescription code	Composition (I)
			1	0.01％TW80	0.01% Tween80 + BAT1406
2	0.1％TW80	0.1% Tween80 + BAT1406
			3	0.2％TW80	0.2% Tween80 + BAT1406
4	0.01％TW20	0.01% Tween 20+ BAT1406
			5	0.1％TW20	0.1% Tween 20+ BAT1406
6	0.2％TW20	0.2% Tween 20+ BAT1406
			7	0.01％PX188	0.01% Poloxamer 188+ BAT1406
8	0.1％PX188	0.1% Poloxamer 188+ BAT1406
			9	0.2％PX188	0.2% Poloxamer 188+ BAT1406
10	KB	BAT1406 (surfactant free)

1. High temperature (40 ℃ C.) test

Taking the above 10 groups of sample formulas, the antibody protein (BAT1406) concentration of 100mg/mL, 80mg/mL, 50mg/mL respectively, subpackaging, performing high temperature test, SEC-HPLC detecting the samples after 1 week (1w) and 2 weeks (2w) of high temperature, comparing with the samples without high temperature (0D), the detecting results are shown in tables 10 a-c:

TABLE 10A SEC-HPLC DETECTION OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (100mg/mL) AT HIGH TEMPERATURE

TABLE 10b SEC-HPLC DETECTION OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (80mg/mL) AT HIGH TEMPERATURE

TABLE 10c SEC-HPLC DETECTION OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (50mg/mL) AT HIGH TEMPERATURE

As can be seen from the data in Table 10, the SEC-HPLC data of the samples containing different concentrations of the BAT1406 antibody after 1 week and 2 weeks of high temperature are all slightly different, the SEC-HPLC monomer purity degradation trends are consistent, and the difference between different surfactants is small.

2. Light (4000lx, 25 ℃ C.) test

10 sets of sample formulations as described in Table 9 were prepared, with antibody protein (BAT1406) concentrations of 100mg/mL, 80mg/mL, 50mg/mL, dispensed, tested in light, and the samples were tested by SEC-HPLC after 5 days (5D) and 10 days (10D) of light, and compared to the samples without light (0D), with the test results as given in tables 11 a-c:

TABLE 11A SEC-HPLC DETECTION DUE OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (100mg/mL) SUITABLE FOR LIGHT

TABLE 11b SEC-HPLC DETECTION OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (80mg/mL) AFTER ILLUMINATION

TABLE 11c SEC-HPLC DETECTION OF PROTEIN SOLUTIONS OF DIFFERENT FORMULATIONS (50mg/mL) AFTER ILLUMINATION

As can be seen from the data in Table 11, the SEC-HPLC data of the samples containing different concentrations of the BAT1406 antibody after 5 days and 10 days of illumination are not very different, the SEC-HPLC monomer purity degradation trends are consistent, and the difference between different surfactants is small.

3. Limiting dilution

Respectively taking protein solutions with the same volume and containing surfactants with different concentrations, wherein the concentrations of antibody proteins (BAT1406) are respectively 100mg/mL, 80mg/mL and 50mg/mL, adding the protein solutions into 0.9% NaCl solution (normal saline) according to the proportion (100 mu L of sample: 20mL of 0.9% sodium chloride solution), slightly and fully mixing the mixture uniformly, observing the properties of the mixture, and detecting insoluble particles of the protein solution after the mixture is placed for 2 hours.

The detection method of the insoluble particles comprises the following steps: according to the general rule 0903 of pharmacopoeia 2015 fourth edition of the people's republic of China: insoluble microparticle inspection method. After the instrument is cleaned to be qualified, 4 test sample contents are carefully combined in a clean workbench, placed in a sampling cup, kept stand for 2 minutes to remove bubbles, and placed on a sampler. Stirring was started to mix the solution evenly (avoiding bubbles), and the amount of sample was 4.0ml each time, measured 4 times by the method. The first measurement data is discarded, the average value of the subsequent 3 measurement data is taken as the measurement result, and the number of particles contained in each container is calculated according to the sampling volume and the volume of the marking device of each container.

The results are shown in tables 12a-c, FIG. 8.

TABLE 12a Change in microparticles of 100mg/ml protein solution in physiological saline

TABLE 12b Change of microparticles in 80mg/ml protein solution in physiological saline

TABLE 12c Change of microparticles in 50mg/ml protein solution in physiological saline

The mean data of the particles in table 12 are plotted to give figure 8. As can be seen from fig. 8, the number of microparticles was much higher in the surfactant-free sample (KB group) than in the other groups, the number of microparticles was significantly reduced in the sample to which the surfactant was added at a level of 0.01%, and when the surfactant concentration was increased to 0.1%, the number of microparticles was reduced to the same level as that in the physiological saline, and the concentration was further increased to 0.2%, the number of microparticles was not greatly changed in the sample, and the surfactant content was not preferably large. The difference between the 3 surfactants is also small.

Referring to the types and content of surfactants in commercially available monoclonal antibody preparations, preferably Tween80, the content is determined to be 0.1%.

Example 5: effect of different adjuvant formulations on antibody stability

Prescription study-1 (40 ℃ high temperature stability experiment): the specific composition and content are shown in Table 13 (wherein, the content of antibody BAT1406 is 100mg/mL, 80mg/mL, 50mg/mL, respectively). The SEC main peak, IEC main peak and acid region were analyzed after standing at 40 ℃ for 0 day, 1 week, 3 weeks and 4 weeks.

As can be seen from the plot of the SEC main peak data (FIGS. 9a-c), the SEC monomer purity stability of samples No.1, 7, 8, 9, 12, 14, and 15 is better; as can be seen from the IEC main peak data plot (FIGS. 10a-c), the IEC main peak content stability of samples No.1, 3, 4, 9, and 15 is better; as can be seen from the IEC acid region data plots (FIGS. 10d-f), the IEC acid region contents of samples Nos. 1, 2, 3, 4, and 10 were more stable.

Prescription study-2 (4000lx light stability experiment): the specific composition and content are shown in Table 13 (wherein, the content of antibody BAT1406 is 100mg/mL, 80mg/mL, 50mg/mL, respectively). The main SEC peak and IEC peak and acid region were analyzed at day 0, day 7 and day 14 under 4000lx illumination.

From the plot of the SEC main peak data (FIGS. 11a-c), the SEC main peak content was greatly reduced for formulas No. 8, 9, 12, and 15; from the IEC main peak data plots (FIGS. 12a-c), the IEC main peak content for

formulas

5, 6, 8, 9, 14, and 15 decreased significantly; from the IEC acid region data plots (FIGS. 12d-f), the IEC acid region contents of formulas Nos. 1, 8, 9, 12, and 15 increased faster.

TABLE 13 composition and content of the ingredients of the different prescriptions of excipients

Note: nos. 1 to 15 in Table 13 correspond to Nos. A170112-1 to A170112-15 in the drawings, respectively.

Prescription screening study-3 (pH): for 3 stabilizers: arginine hydrochloride, trehalose, and sorbitol, (wherein the content of antibody BAT1406 was 100mg/mL, 80mg/mL, and 50mg/mL, respectively, and the remaining components of the screening system are shown in Table 14). Analyzing the conditions of an SEC main peak, an IEC main peak and an acid region after standing at the temperature of 40 ℃ for 0 day, 1 week, 3 weeks and 4 weeks within the pH range of 4.6-5.8; the SEC main peak, the IEC main peak and the acid region were analyzed after standing for 0 day, 1 week and 2 weeks under the illumination of 4000lx, and the analysis results are shown in FIGS. 13 to 16.

Under the condition of 40 ℃, SEC data analysis shows that the SEC monomer purity of 3 groups of stabilizer samples is reduced along with the reduction of the pH value of the samples, wherein the samples of arginine hydrochloride (Arg-HCl) group are greatly influenced by the pH value, and the samples of trehalose (Tre) group are slightly influenced by the pH value. On an overall level, trehalose (Tre) is superior to sorbitol (Sor) to arginine hydrochloride (Arg-HCl) in terms of SEC monomer purity. Wherein the SEC monomer purity of the arginine hydrochloride (Arg-HCl)5.67 and arginine hydrochloride (Arg-HCl)5.44 samples was close to that of trehalose (Tre) and sorbitol (Sor), and the results are shown in FIGS. 13 a-c; according to IEC main peak data analysis, the IEC main peak content of 3 groups of stabilizer samples is reduced along with the reduction of the pH value of the samples, wherein arginine hydrochloride (Arg-HCl) group samples are greatly influenced by the pH value, and trehalose (Tre) and sorbitol (Sor) group samples are slightly influenced by the pH value. On the whole level, the content of the IEC main peak is equivalent to that of sorbitol (Sor) and is superior to arginine hydrochloride (Arg-HCl). The IEC main peak content of the samples of the arginine hydrochloride (Arg-HCl)5.67 and arginine hydrochloride (Arg-HCl)5.44 groups was significantly better than that of the trehalose (Tre) and sorbitol (Sor) groups, and the results are shown in FIGS. 14 a-c; as can be seen from the analysis of the IEC acid region data, the IEC acid region content of all the 3 groups of stabilizer samples decreases with the decrease of the pH of the samples, wherein the samples of the arginine hydrochloride (Arg-HCl) group are greatly influenced by the pH, and the samples of the trehalose (Tre) and sorbitol (Sor) group are slightly influenced by the pH. On the whole level, arginine hydrochloride (Arg-HCl) was superior to trehalose (Tre) and sorbitol (Sor) groups in terms of IEC acid region content, and the results are shown in FIGS. 14 d-f.

Under the condition of illumination of 4000lx, the SEC data analysis shows that the SEC stability of the samples of the arginine hydrochloride (Arg-HCl) group is superior to that of the sorbitol (Sor) group and the trehalose (Tre) group on the whole due to the performances of the 3 stabilizers; and 3 groups of stabilizers have better stability under the condition of slightly low pH, and the results are shown in FIGS. 15 a-c; from the analysis of the IEC main peak data, it can be seen that the IEC stability of the samples of the arginine hydrochloride (Arg-HCl) group is superior to that of the sorbitol (Sor) group and trehalose (Tre) group as a whole in the analysis of 3 stabilizers, and the results are shown in FIGS. 16 a-c; as can be seen from the analysis of IEC acid region data, the IEC stability of the arginine hydrochloride (Arg-HCl) group samples is superior to that of the sorbitol (Sor) group and the trehalose (Tre) group on the whole in the performances of 3 stabilizers; at pH < 5.4, the 3 groups of stabilizers performed less differently at acid zone levels, and the results are shown in FIGS. 16 d-f.

In conclusion, the samples in the arginine hydrochloride (Arg-HCl) group are influenced by pH more greatly and the samples in the trehalose (Tre) group are influenced by pH less under the condition of high temperature (40 ℃). On the whole level, trehalose (Tre) is superior to sorbitol (Sor) and arginine hydrochloride (Arg-HCl) in the SEC monomer purity and IEC main peak content; in terms of IEC acid region content, arginine hydrochloride (Arg-HCl) is superior to trehalose (Tre) and sorbitol (Sor) groups. The SEC monomer purity of arginine hydrochloride (Arg-HCl)5.67 group samples and arginine hydrochloride (Arg-HCl)5.44 group samples is close to that of trehalose (Tre) group and sorbitol (Sor) group samples, and the IEC main peak content of the arginine hydrochloride (Arg-HCl)5.67 group samples and the arginine hydrochloride (Arg-HCl)5.44 group samples is obviously superior to that of the trehalose (Tre) group samples and the sorbitol (Sor) group samples. Under the condition of illumination (4000lx), the SEC monomer purity and IEC main peak content stability of the arginine hydrochloride (Arg-HCl) group sample are superior to those of a sorbitol (Sor) group and a trehalose (Tre) group on the whole; and 3 groups of stabilizers have better stability under the condition of slightly low pH. When the pH is less than 5.4, 3 groups of stabilizing agents have little difference in the performance of acid region content, so that 3 groups of optimal formulation combinations can be screened, and the table 14 is specifically seen.

Of these, arginine hydrochloride (Arg-HCl) is the most prominent among the overall stability, especially arginine hydrochloride (Arg-HCl) group 5.44. At present, the stabilizers of the monoclonal antibody preparations on the market mostly adopt polyhydric alcohols and saccharides, and the experimental results show that the IEC-HPLC acid region content of arginine hydrochloride (Arg-HCl) group samples is lower than that of the polyhydric alcohols and the saccharides under the condition of high temperature (40 ℃), which indicates that the prescription has more advantages in the aspect of slowing down the increase of the acid region of the samples; in addition, under the condition of illumination (4000lx), the SEC monomer purity and IEC main peak content stability of the arginine hydrochloride (Arg-HCl) group sample are also superior to those of polyhydric alcohol and saccharide on the whole.

Note: the arginine hydrochloride group in this example refers to a formulation in which the stabilizing agent is 3.1% arginine hydrochloride + 0.1% methionine.

TABLE 14 optimal formulation formula combinations

Example 6: study of biological Activity

Taking L-929 of logarithmic phase, inoculating in 96-well plate, 8000 cells/hole/100 ul. day 2, diluting 1406 antibody, hIgG negative control group (initial concentration is 750ng/ml, diluting with 1/3 dilution concentration downwards, setting 2 multiple holes for each concentration, setting up 8 gradients) with culture medium containing 10% FBS and rhTNF- α (concentration 1ng/ml), mixing, incubating at room temperature for 30 min, adding 5000ng/ml actinomycin D, making its concentration reach 200ng/ml, mixing, adding the above mixture into L-929 cell, 1ng/ml00 ul/well (final volume 200 ul/well, half final concentration for antibody, rhTNF- α and actinomycin D), 37 ℃/5% CO₂The culture was carried out in an incubator for 24 hours. After 24 hours, the plate was shaken off the medium, 100 ul/well fresh medium was added, 10 ul/well CCK-8 was added, 37 deg.C, 5% CO₂The incubation was continued for 4 hours in the incubator and the OD450 read. The EC50 of the antibody was calculated 1406.

Room temperature stability study: BAT1406 antibody preparations (A1, B1, C1, A2, B2, C2, A3, B3 and C3 preparations, the specific components are shown in Table 1) are stored at room temperature, an in vitro TNF neutralization test (L-929 bioactivity detection) is carried out, and the stability research time is 4 weeks. In vitro neutralization TNF test results show that when nine formulations A1, B1, C1, A2, B2, C2, A3, B3 and C3 are used on day 0, EC50 is 19.395ng/ml, 20.489ng/ml, 20.565ng/ml, 21.806ng/ml, 21.856ng/ml, 19.722ng/ml, 23.585ng/ml, 21.448ng/ml and 19.922ng/ml respectively, and after being placed at room temperature for 4 weeks, EC50 is 20.465ng/ml, 20.385ng/ml, 20.921ng/ml, 20.817ng/ml, 21.812ng/ml, 20.934ng/ml, 20.227ng/ml, 20.583ng/ml and 20.33ng/ml respectively, the biological activity of the formulations is not influenced, and specific results are shown in FIGS. 17 a-i.

Long-term stability study (2-8 ℃): BAT1406 antibody preparations (A1, B1, C1, A2, B2, C2, A3, B3, C3 preparations) were stored at 2-8 ℃ for a stability study period of 36 months. Samples are taken at 3 rd month, 6 th month, 12 th month, 18 th month, 24 th month, 30 th month and 36 th month respectively to carry out an in vitro TNF neutralization test (L-929 bioactivity test), and the results show that the bioactivity is basically unchanged at 0 th month, 3 rd month, 6 th month, 12 th month, 18 th month, 24 th month, 30 th month and 36 th month, which indicates that the preparations of A1, B1, C1, A2, B2, C2, A3, B3 and C3 can keep stable for at least 36 months at 2-8 ℃, and the specific results are shown in Table 15.

TABLE 15a 36 month stability study of three formulations of BAT1406(100mg/ml) at 2-8 deg.C

TABLE 15b 36-month stability Studies of three formulations (80mg/ml) of BAT1406 at 2-8 deg.C

TABLE 15c 36 month stability Studies of three formulations (50mg/ml) of BAT1406 at 2-8 deg.C

Example 7: repeated freeze-thaw study of formulations

Freeze-thaw study-1 preparation of BAT1406 antibody preparation (A, B, C preparation) into 3 samples (specific components are shown in Table 1) with 3 concentrations, namely a low concentration sample LOQ (50mg/mL), a medium concentration sample MOQ (80mg/mL) and a high concentration sample HOQ (100mg/mL), respectively, taking out from a refrigerator at-80 ℃, standing at room temperature for more than 2h, thawing and then freezing, and repeating for 5 times, wherein the freezing time between two thawing is not less than 12 h. And (4) measuring the concentration value of the quality control after freeze thawing by multiple holes, comparing the measured value with the theoretical value, and inspecting the stability of the sample after 5 times of freeze thawing. Each sample was diluted 10 ten thousand times separately for Elisa testing, and the recovery and precision of the duplicate well assay are shown in Table 16.

TABLE 16 Freeze-thaw experimental results of three preparations of recombinant human anti-TNF- α monoclonal antibody BAT1406

The result shows that after 5 times of freeze thawing of samples with three concentrations of the BAT1406 antibody preparation (A preparation), the recovery rate ranges from 99.6 to 109.4 percent, the precision ranges from 0.8 to 4.8 percent, after 5 times of freeze thawing of samples with three concentrations of the BAT1406 antibody preparation (B preparation), the recovery rate ranges from 100.2 to 110.7 percent, the precision ranges from 0.6 to 6.7 percent, after 5 times of freeze thawing of samples with three concentrations of the BAT1406 antibody preparation (C preparation), the recovery rate ranges from 98.4 to 107.9 percent, the precision ranges from 0.5 to 5.8 percent, and the stability of the antibody preparation meets the experimental requirements under the freeze-thawing condition of low, medium and high concentrations and 5 times of repeated freeze thawing. It is noteworthy that the recovery rate of the antibody can still reach more than 98% after the three formulations are subjected to repeated freeze thawing 5 times at a high concentration of 100mg/mL, A, B, C, which is not obvious, because the antibody formulation is generally more unstable at the high concentration, and the high concentration antibody formulation may be accompanied by negative effects of antibody aggregation, reduced solubility, reduced stability and the like.

Freeze-thaw study-2 pharmaceutical formulations (A1, B1, C1, A2, B2, C2, A3, B3, C3 formulations) were cycled 5 times from a frozen state to a liquid state, and freeze-thaw performance of BAT1406 antibody drugs at protein concentrations of 100mg/ml, 80mg/ml, 50mg/ml was evaluated. The results of the experiments in which the formulations (a1, B1, C1, a2, B2, C2, A3, B3, C3 formulations) were evaluated for the effect of 5 rapid and slow cold/thaw cycles starting at-80 ℃ or-20 ℃, respectively, are shown in table 17. The results indicate that the BAT1406 antibody pharmaceutical formulations can be thawed/frozen at least 5 times without any detrimental effect on physicochemical properties or biological activity. The results also show that there are a small number of particles that are only visible under the microscope, whether fast or slow or fast freeze/thaw cycles; in vitro neutralization of TNF also showed that its biological activity was unaffected.

TABLE 17a results of a preparation freeze/thaw experiment of recombinant human anti-TNF- α monoclonal antibody BAT1406(100mg/mL)

TABLE 17b results of freeze/thaw experiments with recombinant human anti-TNF- α monoclonal antibody BAT1406(80mg/mL)

TABLE 17c results of freeze/thaw experiments for recombinant human anti-TNF- α monoclonal antibody BAT1406(50mg/mL)

Example 8: study of microorganisms

Microbiological studies of pharmaceutical formulations (a1, B1, C1, a2, B2, C2, A3, B3, C3 formulations) are required to determine whether the formulations can support microbial growth. The sterile preparation was inoculated directly at low levels (NMT100cfu/ml) by inoculation with microorganisms (e.g. Staphylococcus aureus, ATDD-No.:6538p, Candida albicans, ATDD-No.:10231, Aspergillus niger, ATDD-No.:16404, environmental isolate) and the inoculated preparation was then examined for overall microbial growth. The indicators evaluated were mainly the number of micro-organisms under the microscope and the change in turbidity, wherein the lack of turbidity is an indicator of no overall growth and was detected in the inoculated container after 14 days. In addition, the microorganisms cannot be reisolated from these containers. Table 18 shows that the formulation does not support microbial growth if stored at room temperature 20-25 ℃ for 14 days.

TABLE 18a microbiological assay of three formulations of the recombinant human anti-TNF- α monoclonal antibody BAT1406(100mg/mL)

TABLE 18b microbiological assay of three formulations of the recombinant human anti-TNF- α monoclonal antibody BAT1406(80mg/mL)

TABLE 18c microbiological assay of three formulations of recombinant human anti-TNF- α monoclonal antibody BAT1406(50mg/mL)

-: turbidity is not changed

Example 9: subcutaneous injection of in vivo metabolic conditions

The subcutaneous injection of preparations A1, B1, C1, A2, B2, C2, A3, B3, C3 was performed to SD rats, respectively, and the drug metabolism of the three preparations in SD rats was compared.A weight of the animal (average weight 100 g/mouse) was weighed, and an amount of antibody required for each rat was calculated at 32mg/kg, and 3.2mg of antibody was injected to each rat (3 SD rats per administration, combined with 27 rats), first, 0h of serum was taken from SD rats, frozen at-80 ℃ for use, after the injection of the antibody, blood samples were taken from the tails of rats, respectively, at 1h, 4h, 24h, 48h, 72h, 96h, 7day, 9day, 11day, 13day, SD 15day, 22day, 29day, 36day, 54day, and separated serum was stored at-80 ℃ for use, then, the antibody concentration in blood was measured, and the in vivo data were analyzed by ELISA to verify that the drug metabolism of the rats satisfied the respective subcutaneous injection of the drug metabolism of the rat, and the antibody metabolism of the rat was observed for nine days, and the clinical conditions of TNF-18. A mouse, TNF-14C preparation, showing that the recombinant protein preparation was able to be consistent with the subcutaneous injection of the mouse.

Example 10 recombinant human anti-TNF- α monoclonal antibody BAT1406 protein sequence

A human antibody for treating the disease associated with TNF- α is the recombinant human anti-TNF- α monoclonal antibody BAT1406, which is expressed in CHO cell by gene engineering means and purified by a series of standard chromatographic steps, BAT1406 is IgG antibody with molecular weight of 148kDa and is composed of 2 pieces of IgG1^z,aHeavy chain and 2 kappa light chains. Each heavy chain contains 451 amino acids, has the molecular weight of 49kDa, and has the amino acid sequence shown as SEQ ID NO. 1; each light chain contains 214 amino acids, has a molecular weight of 24kDa, and has an amino acid sequence shown as SEQ ID NO. 2.

Heavy chain amino acid sequence of recombinant human anti-TNF- α monoclonal antibody BAT1406

SEQ ID NO.1

>BAT1406-heavy chain-451aa

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Recombinant human anti-TNF- α monoclonal antibody BAT1406 light chain amino acid sequence

SEQ ID NO.2

>BAT1406-light chain-214aa

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

Example 11 expression and purification of recombinant human anti-TNF- α monoclonal antibody BAT1406

According to the method of Wood et al, J Immunol.145:3011(1990) et al, monoclonal antibody anti-TNF- α antibody specifically binding to TNF- α is expressed in CHO cells expression vectors containing antibody genes were constructed by a conventional Molecular biology method (Molecular Cloning) using a cell line derived from CHO-k1 cells (ATCC CCL61) as a host cell expression procedure for a high yield stable cell line is briefly described as follows, host cells in logarithmic growth phase are grown in CD-CHO medium (Gibco, CA), centrifuged and resuspended in fresh CD-CHO medium, and the cell density is counted and adjusted to 1.43X 10⁷Adding 600ul of the cell suspension into an electric shock cup, adding 40ug of linearized plasmid, (sucking with a pipette to mix the cells and the plasmid uniformly, converting by using a Bio-rad electric converter, setting the parameters of the apparatus as the capacitance: 960uFD, the voltage: 300V, and the electric shock time is usually 15-20 milliseconds to be normal), and adding the cell suspension after electric shock into the electric shock cupThe cells were immediately resuspended in CD-CHO medium preheated at 37 ℃ and 100ul per well in 96-well plates, and 2-3 days later equal amounts of selection medium (CD-CHO media +50uM MSX) were added. The expression level of the antibody was determined by assaying the cell culture supernatant of 96-well plates. Clones with higher expression levels were transferred from 96-well plates to 24-well plates, and when cells grew to a certain number, cells were transferred to 6-well plates so that 5ml of medium per well contained 2X 10⁵Individual cells, cells were assayed for antibody production and yield. Typically 20-30 clones were transferred to shake flasks for further evaluation. Finally, 5-8 clones with the highest expression level are selected for subcloning and further expression detection. The feed liquid is harvested, the cells and the culture medium are separated by low-speed centrifugation, and the centrifuged supernatant is further clarified by high-speed centrifugation. Affinity purification and ion exchange purification with protein A, using media like Mab Select SuRe and Capto S from GE.

Example 12: specificity study of recombinant antibody BAT1406

Pharmacological research mainly includes that the recombinant human anti-TNF- α monoclonal antibody is subjected to in vitro pharmacodynamic test, the in vitro test mainly includes pharmacodynamic detection of the binding capacity with TNF, the antibody specificity, the competitiveness of TNF binding with a receptor, the inhibition effect of TNF- α biological activity, a cell toxicity test and the like, the specific binding capacity of the recombinant human anti-TNF- α monoclonal antibody and TNF- α is compared by taking an antibody specificity as an example, the specific test method includes that firstly, the rhTNF- α, the rhTNF- β or the rmTNF- α are diluted into 100ng/100 mul by PBS, the diluted rhTNF- α, rhTNF- β or the rmTNF- α are added into a 96-well enzyme-labeled plate, 100 mul/well, overnight at 4 ℃, the daily blocking with 3% is carried out, 200 mul/well is carried out, after the supernatant is left at 37 ℃ for 2 hours, the recombinant human anti-TNF-BAT 4 monoclonal antibodies with different concentrations and the recombinant human anti-TNF-BAT 1406 are added into corresponding wells respectively after 100 mul/86525 are added into the 96-well, 100 mul PBS/well, the supernatant is left for overnight at 4 ℃ for overnight, the supernatant is added into the corresponding wells, the recombinant human anti-BAT monoclonal antibodies with different concentrations, the final concentration of 100 mul 1406, the supernatant is added into 100 mul after the supernatant is removed, the supernatant is added into 100 mul supernatant for 100 mul after the serum is added, the serum of 100 mul after the serum is added, the serum of the rTNF-100 mul isThe results remained consistent with the results of specific binding and concentration gradient, but the different concentrations of recombinant human anti-TNF- α monoclonal antibody BAT1406 did not specifically bind to rhTNF- β or rmTNF- α, the negative control human IgG did not specifically bind to rhTNF- α, rhTNF- β or rmTNF- α, demonstrating that BAT1406 only binds to rhTNF- α (IC50 ═ 3X 10-^-9M) without binding to rhTNF- β and rmTNF- α.

Reference documents:

[1]Marc Feldmann,Ravinder N.Maini.Discovery of TNF-αas a therapeutictarget in rheumatoid arthritis preclinical and clinical studies.Joint BoneSpine,2009；69:12-18.

[2]Feldmann M,Brennan FM,Foxwell BMJ,et al.The role of TNF and IL-1in rheumatoid arthritis.Curr Dir Au toimmun,2001；3:188-199.

[3]Daniel Tracey,Lars Klareskog,Eric H.Sssso,et al.Tumor necrosisfactor antagonist mechanisms of action Acomprehensive review.PHarmacology andTherapeutics,2008；117:244-279.

[4]D.J.Shealy,S.Visvanathan.Anti-TNF Antibodies:Lessons from thePast,Roadmap for the Future.Handbook of Experimental PHarmacology 2008；181:101-129.

[5]B.Gatto.Biologics targeted at TNF:design,production andchallenges.Reumatismo,2006；58(2)：94-103.

sequence listing

<110> Baiotai biopharmaceutical GmbH

<120> human antibody preparation for targeted therapy of TNF- α -related diseases

<150>201810142672.4

<151>2018-02-11

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>451

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> > BAT1406-heavy chain-451aaBAT1406 heavy chain

<400>1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210>2

<211>214

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> > BAT1406-light chain-214aaBAT1406 light chain

<400>2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln GlyIle Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValTyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

Claims

1. An antibody preparation characterized by comprising the following components:

(1) 20-120 mg/ml of anti-TNF- α humanized antibody;

(2)5～15mM HAc；

(3)5～15mM His-HCl；

(4) 0.5-10% of a combination of Arg-HCl and Met;

(5)0.01 to 0.2 percent of Tween 80;

(6) water for injection;

the pH value of the preparation is 4.0-7.0;

the anti-TNF- α humanized antibody was the BAT1406 antibody.

2. Antibody preparation according to claim 1, characterized in that it comprises the following components:

(1) 40-120 mg/ml of anti-TNF- α humanized antibody;

(4) 5-15 mM acetic acid;

(5)5～15mM His-HCl；

(4) 0.5-10% of a combination of Arg-HCl and Met;

(5)0.01 to 0.2 percent of Tween 80;

(6) water for injection;

the pH value of the preparation is 4.0-7.0.

3. Antibody preparation according to claim 1, characterized in that it comprises the following components:

(1) 50-100 mg/ml of anti-TNF- α humanized antibody;

(6) 5-10 mM acetic acid;

(7)5～10mM His-HCl；

(4) 0.5-10% of a combination of Arg-HCl and Met;

(5)0.01 to 0.2 percent of Tween 80;

(6) water for injection;

the pH value of the preparation is 4.0-7.0.

4. The formulation of claim 1, wherein:

the combination of the Arg-HCl and the Met is 0.5-5.0% of Arg-HCl and 0.01-2% of Met.

5. The preparation of claim 1, wherein the recombinant human anti-TNF- α monoclonal antibody has a heavy chain represented by SEQ ID NO.1 and a light chain represented by SEQ ID NO. 2.

6. The formulation according to claim 3, characterized by comprising the following components:

(1) 45-120 mg/ml of anti-TNF-alpha human antibody;

(2)5～10mM HAc；

(3)5～10mM His-HCl；

(4)2％～3.5％Arg-HCl；

(5)0.01％～0.5％Met；

(6)0.01％～0.1％Tween 80；

(7) water for injection;

wherein the pH value of the preparation is 5.2-5.6.

7. The formulation according to claim 1, characterized by comprising the following components:

(1) 80-120 mg/ml of anti-TNF-alpha human antibody;

(2)5～10mM HAc；

(3)5～10mM His-HCl；

(4)2％～3.5％Arg-HCl；

(5)0.01％～0.5％Met；

(6)0.01％～0.1％Tween 80；

(7) water for injection;

wherein the pH value of the preparation is 5.2-5.6.

8. The formulation of claim 7, wherein the anti-TNF-alpha human antibody is present in an amount of 85 to 120 mg/ml.

9. The formulation of claim 7, wherein the anti-TNF-alpha human antibody is present in an amount of 90 mg/ml to 110 mg/ml.

10. The formulation according to claim 5, characterized by comprising the following components:

(1)100mg/ml of anti-TNF-alpha human antibody;

(2)5mM HAc；

(3)5mM His-HCl；

(4)3.1％Arg-HCl；

(5)0.1％Met；

(6)0.1％Tween 80；

(7) water for injection;

wherein the pH of the formulation is 5.4 + -0.2.

11. The formulation according to claim 5, characterized by comprising the following components:

(1) anti-TNF-alpha human antibody at 50 mg/ml;

(2)5mM HAc；

(3)5mM His-HCl；

(4)3.1％Arg-HCl；

(5)0.1％Met；

(6)0.1％Tween 80；

(7) water for injection;

wherein the pH of the formulation is 5.4 + -0.2.

12. The formulation according to any one of claims 1 to 11, characterized in that: the osmotic pressure of the preparation is 150-400 mOsm/Kg.

13. The formulation according to any one of claims 1 to 11, characterized in that: the osmotic pressure is 260-340 mOsm/Kg.

14. The formulation according to any one of claims 1 to 11, characterized in that: the formulation also contains NaOH for pH adjustment.

15. The formulation according to any one of claims 1 to 11, characterized in that: the preparation is an injection preparation.

16. The formulation according to any one of claims 1 to 11, characterized in that: the preparation is subcutaneous injection preparation.

17. The formulation according to any one of claims 1 to 11, characterized in that: the formulation remains stable for at least 1 month at room temperature.

18. The formulation of any one of claims 1 to 11, wherein the formulation is stable for at least 36 months at 2 to 8 ℃.

19. The formulation of any one of claims 1-11, wherein said formulation maintains stability after at least 5 cycles of freeze-thawing.

20. The formulation of any one of claims 1-11, wherein the formulation is a pharmaceutical formulation for treating a condition caused by TNF- α.

21. The formulation of claim 20, wherein the condition caused by TNF- α comprises sepsis, an autoimmune disease, an infectious disease, a transplant, a malignancy, a lung disease, an intestinal disease, a heart disease, a cervical joint disease, or a skin disease.

22. The formulation of claim 20, wherein the condition caused by TNF- α comprises rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis.

23. A method of preparing an antibody preparation according to any one of claims 1 to 22, comprising the steps of:

(1) configuring a buffer system comprising a stabilizer;

(2) performing UF/DF ultrafiltration, performing ultrafiltration liquid exchange on the antibody by adopting the buffer solution prepared in the step (1), and then concentrating to the target concentration of the antibody;

(3) adding a surfactant to the liquid prepared in step (2);

(4) filtering, sterilizing, and filling into container.