WO2019065979A1 - ANTI-HUMAN α9 INTEGRIN ANTIBODY-CONTAINING PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

Provided is a stable pharmaceutical composition containing an anti-human α9 integrin antibody that suppresses the production of multimers, degradation products, insoluble impurities, or insoluble microparticles. The pharmaceutical composition contains a pharmaceutically acceptable buffer, and the pH is 5.0-7.0.

Description

Pharmaceutical composition containing anti-human α9 integrin antibody

The present invention relates to a stable pharmaceutical composition comprising an anti-human α9 integrin antibody.

Integrin (integrin) is one of cell surface glycoproteins and functions mainly as adhesion of cells to extracellular matrix (collagen, laminin etc.) and as a receptor for members of the immunoglobulin family (eg ICAM-1, VCAM-1 etc.) Is an adhesion molecule involved in signal transduction from the extracellular matrix. Thereby, the cells receive a signal from the extracellular matrix to induce differentiation, proliferation, cell death and the like. Integrins are heterodimers consisting of two subunits of an α chain and a β chain, and different α chains and β chains exist, there are various combinations, and 24 types of integrin superfamily exist. Integrin knockout mice are lethal or pathological in all subunits, suggesting that individual integrins are required for life maintenance. From this fact, it is thought that integrins that transmit the surrounding environment to cells to promote correspondence function in all aspects of life phenomena and are involved in various pathological conditions.

By the present applicant, the anti-human α9 integrin antibody is useful for diagnosis, prevention or treatment of various diseases involved in the pathogenesis (for example, autoimmune diseases such as rheumatoid arthritis, immune diseases such as allergy and graft rejection) Has been published (Patent Document 1).

On the other hand, in recent years, pharmaceuticals containing various antibodies and other proteins have been developed and are actually provided to medical practice. Many of these pharmaceuticals are dosed by intravenous administration, subcutaneous administration and the like, and thus are provided to medical sites in the form of parenteral pharmaceutical compositions such as liquid preparations or freeze-dried preparations. In particular, since a parenteral pharmaceutical composition is supposed to be directly administered into the body, a stable pharmaceutical preparation is desirable.

In a solution containing a protein such as an antibody, it is desirable to suppress the generation of insoluble fine particles and the like, and from the viewpoint of effectiveness, it is desirable to suppress the generation of a degraded product or the like.

Patent Document 1 discloses that the anti-human α9 integrin antibody itself used in the present invention is heat stable. However, there is no disclosure regarding a stable pharmaceutical composition provided to a medical site. Patent Document 2 discloses a pharmaceutical composition containing anti-IgE antibody E25 and the like. However, there is no disclosure of a stable pharmaceutical composition provided to a medical site comprising an anti-human α9 integrin antibody.

WO 2009/088064 International Publication No. 2004-091658

There is room for further improvement in developing further stable pharmaceutical compositions comprising anti-human α9 integrin antibodies. An object of the present invention is to provide a stable pharmaceutical composition comprising an anti-human α9 integrin antibody.

In particular, the object of the present invention is a stable pharmaceutical composition containing an anti-human α9 integrin antibody, which is formed by, for example, suppressing generation of degradation products or multimers, which are increased by stress such as heat and light. To provide. In addition, another object of the present invention is a stable pharmaceutical composition containing an anti-human α9 integrin antibody, which is formed, for example, by suppressing the formation of insoluble foreign matter or insoluble fine particle which is increased by freezing and thawing, physical vibration or the like. To provide goods.

The present inventors are able to prepare a stable pharmaceutical composition by adjusting the pH of a solution containing an anti-human α9 integrin antibody to a specific range and formulating it (Example 1 described later, etc.) In addition, addition of a specific buffer (Example 2 described later), addition of specific amino acids, salts, sugars, or sugar alcohols (Example 3 described below, etc.), addition of nonionic surfactant It has been found that further stable pharmaceutical compositions can be provided by carrying out (Example 4 and the like described later) and the like, and the present invention has been completed.

That is, the present invention
[1] A pharmaceutical composition comprising an anti-human α9 integrin antibody, a pharmaceutically acceptable buffer, and having a pH of 5.0 to 7.0,
The anti-human α9 integrin antibody is
(1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A pharmaceutical composition selected from the group consisting of antibodies generated by post-translational modification of
[2] The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof [1] of the pharmaceutical composition,
[3] The pharmaceutical composition of [1] or [2], wherein the pharmaceutically acceptable buffer is histidine or a pharmaceutically acceptable salt thereof.
[4] The pharmaceutical composition according to any one of [1] to [3], wherein the concentration of the pharmaceutically acceptable buffer is 1 mmol / L to 300 mmol / L.
[5] The pharmaceutical composition according to any one of [1] to [4], further comprising one or more selected from the group consisting of amino acids, salts, sugars and sugar alcohols.
[6] Amino acids, salts, sugars or sugar alcohols such as methionine, arginine, glycine, lysine and ornithine and pharmaceutically acceptable salts thereof, sodium chloride, sucrose, sorbitol, mannitol, trehalose, xylitol The pharmaceutical composition according to [5], which is one or more selected from the group consisting of erythritol, threitol, inositol, dulcitol, arabitol, isomalt, lactitol, and maltitol.
[7] The pharmaceutical composition of [5] or [6], wherein the amino acids, salts, sugars or sugar alcohols are arginines,
[8] The pharmaceutical composition of any one of [5] to [7], wherein the concentration of the amino acids, salts, sugars or sugar alcohols is 1 mmol / L to 400 mmol / L.
[9] The pharmaceutical composition of any one of [1] to [8], further comprising a nonionic surfactant.
[10] The pharmaceutical composition of [9], wherein the non-ionic surfactant is polysorbate 80 and / or poloxamer 188.
[11] The pharmaceutical composition of [9] or [10], wherein the concentration of the nonionic surfactant is 0.001% (w / v) to 1% (w / v),
[12] The pharmaceutical composition of any one of [1] to [11], wherein the concentration of the anti-human α9 integrin antibody is 1 mg / mL to 1000 mg / mL.
[13] The pharmaceutical composition of any one of [1] to [12], wherein the pharmaceutical composition is a liquid preparation or a lyophilized preparation.
[14] The pharmaceutical composition of any one of [1] to [13], wherein the pharmaceutical composition is a liquid preparation,
[15] A pharmaceutical composition comprising an anti-human α9 integrin antibody, histidine or a pharmaceutically acceptable salt thereof, and having a pH of 5.0 to 7.0, wherein the anti-human α9 integrin antibody is
(1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A pharmaceutical composition selected from the group consisting of antibodies generated by post-translational modification of
[16] The pharmaceutical composition of [1] or [15], wherein the post-translational modification of the anti-human α9 integrin antibody is heavy chain N-terminal pyroglutamylation and heavy chain C-terminal lysine deletion.
[17] A pharmaceutically acceptable buffer, an amino acid, a salt, a sugar or a sugar alcohol as a light stabilizer for producing a stable pharmaceutical composition containing an anti-human α9 integrin antibody Use,
The anti-human α9 integrin antibody is
(1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A use selected from the group consisting of antibodies generated by post-translational modification of
[18] The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof There is a use of [17],
[19] In a pharmaceutical composition containing an anti-human α9 integrin antibody, the degradation product or the amount of the anti-human α9 integrin antibody upon exposure to light by a pharmaceutically acceptable buffer, amino acids, salts, sugar or sugar alcohols A method of suppressing the formation of the body,
The anti-human α9 integrin antibody is
(1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A method selected from the group consisting of antibodies generated by post-translational modification of
[20] The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof There is a way [19],
About.

According to the present invention, a stable pharmaceutical composition comprising an anti-human α9 integrin antibody, in particular, a stable pharmaceutical composition formed by suppressing the formation of a multimer, a degradation product, an insoluble foreign substance or an insoluble microparticle Can provide the goods.

It is a graph which shows the evaluation result (multimeric amount) of the size exclusion chromatography method (SE-HPLC method) before and behind storage of the liquid formulation of various pH performed in Example 1 at 40 ° C. for 1 month. It is a graph which shows the evaluation result (amount of decomposition products) of the SE-HPLC method before and behind storage of the liquid formulation of various pH performed in Example 1 at 40 ° C. for 1 month. It is a graph which shows the evaluation result (impurity (= multimer + decomposition product) amount) of the liquid preparation of various pH performed in Example 1, before and after storage at 40 ° C. for 1 month. It is a graph which shows the evaluation result (the amount of multimers) of the liquid preparation containing various buffer solution components carried out in Example 1 before and after 48 hours of light exposure. It is a graph which shows the evaluation result (the amount of multimers) of the SE-HPLC method before and behind storage of the liquid formulation containing various buffer solution components performed in Example 2 at 40 ° C. for 1 month. It is a graph which shows the evaluation result (amount of decomposition products) of the SE-HPLC method before and behind storage of the liquid formulation containing various buffer solution components performed in Example 2 at 40 ° C. for 1 month. It is a graph which shows the evaluation result (the amount of multimers) of the liquid preparation containing various buffer solution components performed in Example 2, before and after 48 hours of light exposure. It is a graph which shows the evaluation result (the amount of multimers) of the SE-HPLC method of the liquid formulation containing various buffer solution components performed in Example 3 before and after 168 hours of light exposure. It is a graph which shows the evaluation result (multimeric amount) of the SE-HPLC method before and behind storage of the liquid formulation containing arginine as various amino acids in various concentrations at 40 ° C. for 1 month performed in Example 4. It is a graph which shows the evaluation result (multimeric amount) of the SE-HPLC method before and behind storage of the liquid formulation containing sodium chloride as salts in various concentrations at various concentrations for 1 month at 40 ° C. It is a graph which shows the evaluation result (multimeric amount) of the SE-HPLC method before and behind storage of the liquid formulation which contains methionine as amino acids in various concentrations in Example 4 at 40 ° C. for 1 month. It is a graph which shows the evaluation result (multimeric amount) of the SE-HPLC method before and behind storage of the liquid formulation containing sucrose or sorbitol as saccharide | sugar or saccharide alcohol performed in Example 4 at 40 degreeC 1 month. It is a graph which shows the evaluation result (multimeric amount) of the liquid preparation which carried out in Example 4 and which contains arginine or methionine as various amino acids at various concentrations for 48 hours before and after exposure to light. It is a graph which shows the evaluation result (the amount of multimers) of the liquid preparation containing sodium chloride in various concentrations and the liquid preparation containing sucrose or sorbitol carried out in Example 4 before and after exposure for 48 hours. It is a graph which shows the result of having measured the number of insoluble fine particles before and after conducting a shaking test using the liquid preparation which carried out in Example 5 and contains polysorbate 80 as various kinds of nonionic surfactant at various concentrations. It is a graph which shows the result of having measured the number of insoluble fine particles before and behind a freeze-thawing stress test using the liquid preparation which carried out in Example 5 and contains polysorbate 80 as various levels of nonionic surfactant. It is a graph showing the evaluation results (multimeric amount) of the SE-HPLC method before and after storage at 25 ° C. for 1 month of the antibody high concentration liquid preparation containing various histidine, arginine and polysorbate 80 performed in Example 7. is there. It is a graph which shows the evaluation result (amount of decomposition products) of the SE-HPLC method before and behind storage of the antibody high concentration liquid preparation containing various histidine, arginine, and polysorbate 80 performed in Example 7 at 25 ° C. for 1 month. . Evaluation results of the SE-HPLC method before and after storage at 25 ° C. for 1 month of the antibody high concentration liquid preparation containing various histidine, arginine and polysorbate 80 performed in Example 7 (impurity amount (= amount of polymer + It is a graph which shows the amount of decomposition products). It is a graph which shows the evaluation result (the amount of multimers) of SE-HPLC method before and behind storage for the antibody high concentration liquid preparation containing various amino acids, sugar, or sugar alcohol performed in Example 8 at 25 ° C for 1 month. . FIG. 16 is a graph showing the evaluation results (degraded product amounts) of the SE-HPLC method before and after storage of the antibody high concentration liquid preparation containing various amino acids, sugar or sugar alcohol, performed in Example 8 at 25 ° C. for 1 month. Evaluation results of the SE-HPLC method before and after storage at 25 ° C. for 1 month of the antibody high concentration liquid preparation containing various amino acids, sugars or sugar alcohols performed in Example 8 (impurity amount (= amount of polymer + decomposition It is a graph which shows physical quantity). It is a graph which shows the result of having measured the number of insoluble microparticles | fine-particles before and after performing a shaking test using the antibody high concentration liquid formulation containing polysorbate 80 or poloxamer 188 as a nonionic surfactant performed in Example 9. FIG. It is a graph which shows the result of having measured the number of insoluble fine particles before and after performing a freeze thaw stress test using the antibody high concentration liquid preparation containing polysorbate 80 or poloxamer 188 as a nonionic surfactant performed in Example 9 . The SE-HPLC method and the cation exchange chromatography method (CE-HPLC) after storing the antibody high concentration liquid preparation containing various concentrations of histidine, arginine, and poloxamer 188 performed in Example 11 at 25 ° C. for 3 months It is a graph which shows the result (the amount of increase of a multimer) of carrying out the statistical analysis of the evaluation result of method. Statistical analysis of evaluation results of SE-HPLC method and CE-HPLC method after storing the antibody high concentration liquid preparation containing various concentrations of histidine, arginine and poloxamer 188 for 3 months at 25 ° C., which were carried out in Example 11. It is a graph which shows the result (increase amount of decomposition products). Statistical analysis of evaluation results of SE-HPLC method and CE-HPLC method after storing the antibody high concentration liquid preparation containing various concentrations of histidine, arginine and poloxamer 188 for 3 months at 25 ° C., which were carried out in Example 11. It is a graph which shows the result (reduction of the main peak).

As used herein, "stable" means stable to, for example, heat, light, temperature, humidity, shaking, and / or freeze-thaw. For example, it is defined that after storing the pharmaceutical composition under predetermined conditions, the total amount of multimers or degradants contained in the pharmaceutical composition or the increased amount thereof is equal to or less than a specific amount. In certain embodiments, it is defined that the charge analog is below a certain amount. In one embodiment, the insoluble foreign matter is not visually observed after storage of the pharmaceutical composition under predetermined conditions, or the number of insoluble fine particles is equal to or less than a specific number as measured by the light blocking particle counting method described later. Specify.

The “multimer” is a complex formed by collecting anti-human α9 integrin antibodies. The method for measuring multimers is not particularly limited, and, for example, size exclusion chromatography (SE-HPLC), gel electrophoresis, capillary electrophoresis, dynamic light scattering, light blocking particle counting, A microflow imaging method and the like are included, and an embodiment is a SE-HPLC method. In the SE-HPLC method, the area of the detected peak is measured by an automatic analysis method and divided by the sum of all peak areas including the main peak to calculate as a percentage (%), the amount of each multimer I assume. The main peak means a peak of the active substance. Peaks having a retention time shorter than that of the main peak of SE-HPLC are combined to obtain the total amount of multimers (hereinafter referred to as the amount of multimers). The amount of the multimer is 10% or less in an embodiment, 5% or less in an embodiment, and 3% or less in an embodiment. In addition, the amount of increase in amount of multimer (the difference between the amount of multimer after storage for a specific period under a specific condition and the amount of multimer at the start of test (storage)) is 5% or less as an aspect, an aspect As 2% or less, in one embodiment as 1% or less, and in one embodiment as 0.5% or less. The multimeric amount or the increased amount of the multimeric amount can be optionally combined with each storage condition described in the definition of “stable pharmaceutical composition” described later, if desired.

The “degradant” is a fragment formed by partial detachment of an anti-human α9 integrin antibody. The method for measuring the decomposition product is not particularly limited, and includes, for example, the SE-HPLC method, gel electrophoresis, capillary electrophoresis and the like, and in one embodiment, the SE-HPLC method. The method of measuring the decomposition product by the SE-HPLC method is carried out in the same manner as the measurement of the multimer by the above-mentioned SE-HPLC method. In addition, peaks having a retention time longer than that of the main peak of SE-HPLC are combined to obtain the total amount of degradation products (hereinafter referred to as the amount of degradation products). The amount of decomposition product is 10% or less in one embodiment, 5% or less in one embodiment, and 3% or less in one embodiment. In addition, the amount of degradation products (the difference between the amount of degradation products after storage for a specific period under specific conditions and the amount of degradation products at the start of testing (storage)) is 10% or less as an aspect, 5% as an aspect Hereinafter, it is 3% or less as a certain aspect. In addition, about the increase amount of the said decomposition product amount or decomposition product amount, it can combine arbitrarily with each storage conditions described in the definition of the "stable pharmaceutical composition" of the after-mentioned later, if desired.

The "charge analog" is an analog which is recognized in addition to the main peak in analysis by cation exchange chromatography or imaging capillary isoelectric focusing. The method for measuring the charge analog is not particularly limited, and includes, for example, cation exchange chromatography (CE-HPLC), imaging capillary isoelectric focusing (icIEF), etc. Is the CE-HPLC method or the icIEF method. In the CE-HPLC method and the icIEF method, the percentage (%) of the peak is calculated in the same manner as the above-mentioned SE-HPLC method, and is used as the amount of each charge analog. The total amount of charge analog is 0 to 70% in one embodiment, 0 to 50% in one embodiment, 0 to 30% in one embodiment, and 0 to 10% in one embodiment.

The "insoluble foreign matter" can be visually confirmed according to the insoluble foreign matter inspection method for injections of the Japanese Pharmacopoeia.

The “insoluble fine particles” are insoluble complexes formed by collecting anti-human α9 integrin antibodies, pharmaceutical additives and the like. The number of insoluble fine particles can be defined by the light blocking particle counting method as the number of insoluble fine particles is equal to or less than the specific number. In one embodiment, the number of insoluble fine particles of 1.2 μm or more is 1000 / mL or less.

The “stable pharmaceutical composition” is, for example, 3 months at a refrigerated temperature (2 ° C. to 8 ° C.), 6 months as an aspect, 1 year as an aspect, 2 years as an aspect; or room temperature (22 ° C) At 28 ° C., 2 weeks as an embodiment, 1 month as an embodiment, 3 months as an embodiment, 6 months as an embodiment, 1 month as an embodiment; or under accelerated conditions (37 ° C. to 43 ° C.) Or 2 weeks in one embodiment, 4 weeks in one embodiment; or D65 lamp under 1000 lux irradiation, for example, 48 hours, in one embodiment after 168 hours of light exposure, increase amount of multimeric amount or degradable amount, or multimeric amount or degradation It means a pharmaceutical composition whose physical quantity is less than a certain amount.

The anti-human α9 integrin antibody of the following (1) and / or (2) as an anti-human α9 integrin antibody (also referred to as "anti-human α9 integrin antibody used in the present invention") contained in the pharmaceutical composition of the present invention Contains:
(1) An anti-human α9 integrin antibody (WO 2009/088064) comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3
(2) An antibody generated by post-translational modification of the anti-human α9 integrin antibody of (1).

When the antibody is expressed in cells, it is known that the antibody undergoes post-translational modification. Examples of post-translational modification include cleavage of lysine at the heavy chain C terminus with carboxypeptidase, modification to pyroglutamate by pyroglutamylation of heavy chain and light chain N terminus with glutamine or glutamic acid, etc. It is known that heavy chain C-terminal lysine is deleted, and most of heavy chain N-terminal glutamine is modified to pyroglutamate (Journal of Pharmaceutical Sciences, 2008, Vol. 97, p. 2426-2447). It is also known in the art that such post-translational modifications do not affect the activity of antibodies (Analytical Biochemistry, 2006, Vol. 348, p. 24-39).

In one embodiment, the post-translational modification of (2) the anti-human α9 integrin antibody is N-terminal pyroglutamylation of heavy chain. In one embodiment, the anti-human α9 integrin antibody of (2) comprises a heavy chain consisting of the amino acid sequence of SEQ ID NO: 1 in which glutamine at amino acid No. 1 is modified to pyroglutamate and the amino acid sequence shown in SEQ ID NO: 3 Anti-human α9 integrin antibody comprising a light chain

In one embodiment, the post-translational modification of (2) the anti-human α9 integrin antibody is deletion of a lysine at the C-terminus of the heavy chain. In one embodiment, the (2) anti-human α9 integrin antibody comprises a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 449 of SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 , Anti-human α9 integrin antibody.

In one embodiment, the post-translational modification of the anti-human α9 integrin antibody of (2) is pyroglutamylation at the N-terminus of heavy chain and deletion of lysine at the C-terminus of heavy chain. In one embodiment, the anti-human α9 integrin antibody of (2) is a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 449 of SEQ ID NO: 1 in which glutamine at amino acid number 1 is modified to pyroglutamate and It is an anti-human α9 integrin antibody comprising a light chain consisting of the amino acid sequence shown in 3.

The humanized anti-human α9 integrin antibodies used in the present invention also include anti-human α9 integrin antibodies produced by culturing the following host cells:
Transformation with an expression vector containing a polynucleotide comprising a base sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a polynucleotide comprising a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3 The host cell or
An expression vector comprising a polynucleotide comprising a nucleotide sequence encoding a heavy chain consisting of an amino acid sequence as shown in SEQ ID NO: 1 and an expression comprising a polynucleotide comprising a nucleotide sequence encoding a light chain consisting of an amino acid sequence as shown in SEQ ID NO: 3 Host cell transformed with vector.

The anti-human α9 integrin antibody used in the present invention can be easily produced by those skilled in the art using methods known in the art based on the sequence information of the anti-human α9 integrin antibody disclosed herein. . As a method for producing the anti-human α9 integrin antibody used in the present invention, the method disclosed in WO 2009/088064 can be mentioned.

The amount of the anti-human α9 integrin antibody used in the present invention in one unit pharmaceutical composition (formulation) includes, for example, 1 mg to 1000 mg, and in one embodiment 1 mg to 500 mg, in one embodiment 1 mg to 30 mg And 10 mg to 200 mg in one embodiment, 100 mg to 250 mg in one embodiment, 50 mg to 250 mg in one embodiment, and 100 mg to 200 mg in one embodiment. The above upper limit and the lower limit can be optionally combined as desired.

When the pharmaceutical composition is in a solid state (eg, lyophilized formulation, spray-dried formulation, etc.), the amount of anti-human α9 integrin antibody used in the present invention is, for example, 1 mg to 1000 mg, and in one embodiment 1 mg to 500 mg, in one embodiment 1 mg to 30 mg, in one embodiment 10 mg to 200 mg, in one embodiment 100 mg to 250 mg, in one embodiment 50 mg to 250 mg, in one embodiment 100 mg to 200 mg. The above upper limit and the lower limit can be optionally combined as desired.

When the pharmaceutical composition is dissolved at the time of use, the concentration of the anti-human α9 integrin antibody used in the present invention is, for example, 1 mg / mL to 1000 mg / mL, and in one embodiment 1 mg / mL to 500 mg / mL 1 mg / mL to 30 mg / mL in an embodiment, 10 mg / mL to 200 mg / mL in an embodiment, 100 mg / mL to 250 mg / mL in an embodiment, 50 mg / mL to 250 mg / mL in an embodiment, 100 mg / mL to an embodiment It is 200 mg / mL. The above upper limit and the lower limit can be optionally combined as desired.

When the pharmaceutical composition is in a solution state (liquid formulation), examples of the concentration of the anti-human α9 integrin antibody used in the present invention include 1 mg / mL to 1000 mg / mL, and in one embodiment 1 mg / mL to 500 mg / mL. mL, in one embodiment 1 mg / mL to 30 mg / mL, in one embodiment 10 mg / mL to 200 mg / mL, in one embodiment 100 mg / mL to 250 mg / mL, in one embodiment 50 mg / mL to 250 mg / mL, in one embodiment 100 mg / ML to 200 mg / mL. The above upper limit and the lower limit can be optionally combined as desired.

The dose is, for example, 0.002 mg / kg to 100 mg / kg, in one embodiment 0.01 mg / kg to 50 mg / kg, and in one embodiment 1 mg / kg to 30 mg / kg. The above upper limit and the lower limit can be optionally combined as desired.

Indications can include the prevention and / or treatment of various diseases in which integrins are involved, for example, autoimmune diseases such as rheumatoid arthritis, immune diseases such as allergy and graft rejection, and the like.

The “pharmaceutically acceptable buffer” used in the present invention is pharmaceutically acceptable, has a buffering ability in a desired pH range in solution, or has a stabilizing effect. There is no particular limitation if it is.
Specifically, it has a buffering capacity in the following pH range. For example, 5.0 to 7.0, an embodiment 5.5 to 6.5, an embodiment 5.6 to 6.2, an embodiment 6.0, an embodiment 5.8. The above upper limit and the lower limit can be optionally combined as desired.
In the present specification, the pH of the pharmaceutical composition means, in the case of a liquid preparation, the pH in a solution state, and in the case of a non-liquid preparation (for example, a freeze-dried preparation, a spray-dried preparation, etc.) It is dissolved in a solvent (for example, water for injection) to mean pH in a solution state.

As the buffer component, for example, acetic acid, citric acid, phosphoric acid, histidine, pharmaceutically acceptable salts thereof and the like can be mentioned. One embodiment is histidine or a pharmaceutically acceptable salt thereof and the like, and one embodiment is histidine.
Acetic acid, citric acid, phosphoric acid, and histidine, and their pharmaceutically acceptable salts do not affect the stability of the anti-human α9 integrin antibody used in the present invention, as well as as a buffer component. Or show the effect of improving the stability.
Also, acetic acid, citric acid, phosphoric acid, histidine, or their pharmaceutically acceptable salts inhibit the formation of degradation products or multimers of the humanized anti-human α9 integrin antibody used in the present invention, particularly by exposure to light. Light stabilization effect.

The buffer component can be used singly or in appropriate amounts as appropriate.
The concentration of the buffer is not particularly limited as long as the pH can be adjusted within the desired pH range. The concentration of the buffer is, for example, 1 mmol / L to 300 mmol / L, in one embodiment 10 mmol / L to 150 mmol / L, in one embodiment 1 mmol in the case of a solution (liquid formulation) dissolved in a solvent (eg water for injection) / L to 50 mmol / L, and in one embodiment 5 mmol / L to 175 mmol / L. The above upper limit and the lower limit can be optionally combined as desired.

The amino acids, salts, sugars or sugar alcohols used in the present invention are pharmaceutically acceptable and do not affect the stability or improve the stability of the anti-human α9 integrin antibody used in the present invention If it is a thing, it will not be restrict | limited in particular.

Examples of amino acids include methionine, arginine, glycine, lysine, ornithine, or pharmaceutically acceptable salts thereof, and the like. In one embodiment, methionine, arginine, and glycine, and in another embodiment, arginine.
As a salt, sodium chloride etc. can be mentioned, for example.
Examples of sugars or sugar alcohols include sucrose, sorbitol, mannitol, trehalose, xylitol, erythritol, threitol, inositol, dulcitol, arabitol, isomalt, lactitol, maltitol and the like. Some embodiments are sucrose and sorbitol, and one embodiment is sorbitol.
The amino acids, salts, sugars or sugar alcohols are, in one aspect, arginine, methionine, or a pharmaceutically acceptable salt thereof, sorbitol, sucrose, sodium chloride, and in one aspect, arginine.

One or more amino acids, salts, sugars or sugar alcohols can be used appropriately.
The types and combinations of amino acids, salts, sugars or sugar alcohols are not particularly limited as long as they do not affect the stability of the anti-human α9 integrin antibody used in the present invention or improve the stability. I will not.
Among the anti-human α9 integrin antibodies used in the present invention, arginine, methionine, sorbitol, sucrose and sodium chloride particularly exhibit an effect on light stabilization.
The amount of amino acids, salts, sugars or sugar alcohols is not particularly limited as long as it does not affect the stability or improve the stability of the anti-human α9 integrin antibody used in the present invention.
For example, the concentration in the case of solution (liquid preparation) dissolved by a solvent is 1 mmol / L to 400 mmol / L, in one embodiment 1 mmol / L to 300 mmol / L, in one embodiment 100 mmol / L to 300 mmol / L in one embodiment And 100 mmol / L to 200 mmol / L. The above upper limit and the lower limit can be optionally combined as desired.

As a light stabilizer of the anti-human α9 integrin antibody used in the present invention, pharmaceutically acceptable buffers, amino acids, salts, sugars or sugar alcohols are used. For example, acetic acid, citric acid, phosphoric acid, histidine, arginine, methionine, or pharmaceutically acceptable salts thereof, sorbitol, sucrose, sodium chloride and the like can be mentioned.

The nonionic surfactant used in the present invention is not particularly limited as long as it is pharmaceutically acceptable and has a stabilizing effect on the anti-human α9 integrin antibody used in the present invention.

Specifically, the non-ionic surfactant includes, for example, sorbitan monocaprylate, sorbitan monolaurate, and sorbitan fatty acid esters such as sorbitan monopalmitate; glycerol monocaprylate glycerol, glycerol monomyristate glycerol, and monostearate acid Glycerin fatty acid esters such as glycerol; polyglycerol fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polystearate monoesterate Oxyethylene sorbitan, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and poly tristearate Polyoxyethylene sorbitan fatty acid ester such as xylethylene sorbitan; polyoxyethylene sorbitol fatty acid ester such as polyoxyethylene sorbitol tetrastearate and polyoxyethylene sorbitol polytetraoleate; polyoxyethylene glycerin fatty acid such as polyoxyethylene glyceryl monostearate Esters; polyethylene glycol fatty acid esters such as polyethylene glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, and polyoxyethylene polyoxyethylene Polyoxyethylen such as propylene cetyl ether Polyoxypropylene alkyl ether; Polyoxyethylene alkyl phenyl ether such as polyoxyethylene nonyl phenyl ether; Polyoxyethylene hydrogenated castor oil such as polyoxyethylene castor oil and polyoxyethylene hydrogenated castor oil (polyoxyethylene hydrogenated castor oil) Polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; 6 to 18 Hydrophilic-Lipophilic Balances such as polyoxyethylene fatty acid amides, such as polyoxyethylene octadecanamide (HLB) containing surfactant.

The nonionic surfactant can be used alone or in combination of two or more.

In one embodiment, polyoxyethylene sorbitan fatty acid ester or polyoxyethylene polyoxypropylene alkyl ether, and in one embodiment polysorbate 20, 21, 40, 60, 65, 80, 81, or 85, and a pluronic surfactant It is polysorbate 20 and 80 in one embodiment, or poloxamer (poloxamer 188), polysorbate 80 in one embodiment, and poloxamer (poloxamer 188) in one embodiment.

The nonionic surfactant has an action to suppress the formation of insoluble foreign matter and / or insoluble fine particles, etc., and is within the range and / or stability for stabilizing the anti-human α9 integrin antibody used in the present invention. To the extent that it does not affect the
The amount of non-ionic surfactant is not particularly limited as long as it has a stabilizing effect on the anti-human α9 integrin antibody used in the present invention.
0.001% (w / v) to 1% (w / v), and in one embodiment 0.01% (w / v) to a solution (liquid formulation) dissolved in a solvent (eg, water for injection) 0.5% (w / v), in one embodiment 0.01% (w / v) to 0.1% (w / v), and in one embodiment 0.01% (w / v) to 0.. It is 03% (w / v). The above upper limit and the lower limit can be optionally combined as desired.

The pharmaceutical composition of the present invention is provided as a liquid preparation by filling the solution in a container, and as a parenteral pharmaceutical composition such as a freeze-dried preparation or a spray-dried preparation by lyophilization or spray drying of the solution. can do. Preferably, it is a liquid formulation.

If necessary, pharmaceutical additives such as suspending agents, solubilizers, preservatives, adsorption inhibitors, sulfur-containing reducing agents, antioxidants and the like can be added to the pharmaceutical composition of the present invention as appropriate.

Examples of suspending agents include methylcellulose, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate and the like.

Examples of the solubilizer include polyoxyethylene hydrogenated castor oil, nicotinic acid amide, polyoxyethylene sorbitan monolaurate, tuna gall, and castor fatty acid ethyl ester.

Examples of the preservative include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, chlorocresol and the like.

Examples of the adsorption inhibitor include human serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hydrogenated castor oil, polyethylene glycol and the like.

The sulfur-containing reducing agent includes, for example, N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, Those having a sulfhydryl group such as a thioalkanoic acid having 1 to 7 carbon atoms can be mentioned.

As the antioxidant, for example, methionine, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbic acid stearate, sulfurous acid Examples thereof include sodium hydrogen, sodium sulfite, triamyl gallate, propyl gallate, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate and the like.

Various pharmaceutical additives can be used appropriately in appropriate amounts within the range that can achieve the desired effects of the present invention. For example, an appropriate amount may be used to adjust the osmotic pressure of the liquid preparation to be 250 to 350 mОsm, which is essentially the same as human blood. Also, for example, when the pharmaceutical composition is in a solution state (liquid formulation), an appropriate amount can be used for the purpose of achieving a viscosity that can be administered. Specifically, when viscosity is measured based on the measurement method described in Example 10 described later, an appropriate amount can be used for the purpose of setting the viscosity to 100 cps or less.

The method for producing the pharmaceutical composition of the present invention comprises the anti-human α9 integrin antibody to be used in the present invention, a pharmaceutically acceptable buffer according to a production method known per se, preferably amino acids, salts, sugars Or a method of further containing a sugar alcohol and / or a nonionic surfactant, and adjusting the pH to 5.0 to 7.0.
In addition, the method for producing the pharmaceutical composition of the present invention comprises the anti-human α9 integrin antibody used in the present invention, histidine or a pharmaceutically acceptable salt thereof according to a production method known per se, and has a pH of 5. Includes methods to adjust from 0 to 7.0.

In the present invention, pharmaceutically acceptable buffers, amino acids, salts, sugars or sugar alcohols for producing a stable pharmaceutical composition containing the anti-human α9 integrin antibody used in the present invention. , As a light stabilizer.
The present invention also includes acetic acid, citric acid, phosphoric acid, and histidine, and arginine, methionine, sucrose, and the like for producing a stable pharmaceutical composition containing the anti-human α9 integrin antibody used in the present invention. Including the use of sodium chloride as a light stabilizer.
"Stable", "stable pharmaceutical composition", "pharmaceutically acceptable buffer", "amino acid, salts, sugar or sugar alcohol" to be used in the present invention as the light stabilizer The description in the pharmaceutical composition of the present invention can be applied as it is.
The description in the pharmaceutical composition of the present invention can be applied as it is to the compounding amount of each component in the use as a light stabilizer of the present invention, the blending method, and the like.
In the use of the present invention as a light stabilizer, acetic acid, citric acid, phosphoric acid, and histidine, and their pharmaceutically acceptable salts, arginine, methionine, sucrose, sodium chloride, as a light stabilizer In providing a pharmaceutical composition containing the anti-human α9 integrin antibody, it is possible to suppress the formation of degradation products or multimers of the anti-human α9 integrin antibody by exposure to light.

In the present invention, in the pharmaceutical composition containing the anti-human α9 integrin antibody used in the present invention, the anti-human by exposure to light with a pharmaceutically acceptable buffer, amino acids, salts, sugar or sugar alcohols. It includes a method of suppressing the formation of degradation products or multimers of α9 integrin antibody.
Furthermore, in the present invention, in the pharmaceutical composition containing the anti-human α9 integrin antibody used in the present invention, acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof, arginine, The method of making methionine, sucrose, sodium chloride suppress the production | generation of the decomposition product or multimer of said anti-human alpha9 integrin antibody by light exposure.
"Pharmaceutically acceptable buffer", "amino acids, salts, sugars or the like" to be used in the method of the present invention for suppressing the formation of degradation products or multimers of the anti-human α9 integrin antibody used in the present invention by exposure to light The description in the pharmaceutical composition of the present invention can be applied as it is to sugar alcohols, "degraded products" and "multimers".
About the compounding quantity of each component in the method of this invention which suppresses the production | generation of the decomposition product or multimer of anti-human alpha9 integrin antibody used for this invention by this invention, the combination method, etc., the said description in the pharmaceutical composition of this invention It can be applied as it is.

The container filled with the pharmaceutical composition of the present invention can be selected according to the purpose of use. For example, it includes one in the form of a prescribed volume such as a vial, an ampoule, and a syringe, and one in the form of a large volume such as a bottle. One embodiment includes a syringe (including a disposable syringe). By pre-filling the syringe with the solution and providing it as a prefilled syringe liquid preparation, in the medical site, the operation such as the dissolution operation becomes unnecessary, and quick response becomes possible.

Examples of the material of the container include glass and plastic. Moreover, as a surface treatment in a container, a silica coating process, a silicone coating process, a sulfur process, various low alkali processes etc. may be given.

Hereinafter, the present invention will be described in more detail by way of reference examples and examples, but the present invention is not limited by these.

Reference Example: Preparation of anti-human α9 integrin antibody
The nucleotide sequence of the polynucleotide encoding the heavy chain of the anti-human α9 integrin antibody used in this example is SEQ ID NO: 2, the amino acid sequence encoded thereby is SEQ ID NO: 1, and the light chain of the anti-human α9 integrin antibody The nucleotide sequence of the encoding polynucleotide is shown in SEQ ID NO: 4, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 3.

According to WO 2009/088064, AG-γ1 (WO 94/20632) into which the heavy chain of the anti-human α9 integrin antibody is inserted and AG- ((WO 94 /) into which the light chain of the anti-human α9 integrin antibody is inserted 20632) was constructed, and an expression plasmid was constructed by linking them. The expression plasmid was expressed using FreeStyle 293 Expression System, and purified antibody was obtained from the culture supernatant using a protein A column. In addition, as a result of analyzing the amino acid modification of the purified anti-human α9 integrin antibody, it was found that pyroglutamylation of the heavy chain N-terminus and deletion of lysine at the heavy chain C-terminal side occurred in most of them.

<< Example 1: Stabilizing effect by selection of optimum pH >>
Preparation solutions (samples No. A-1 to A-7) diluted with the buffer solution shown in Table 1 (sample No. A-1 to A-7) were diluted with 0.22 μm filter so that the anti-human α9 integrin antibody concentration prepared in the reference example was 10 mg / mL. The solution is sterilized by filtration, filled with 1.3 mL each in a glass vial (3 mL volume), stoppered with a rubber stopper, stored for 1 month at 40 ° C. in the upright state, and diluted with the buffer shown in Table 1 Among the formulated solutions, sample no. The exposure was carried out for A-1, 2, 3, 6, 7 with D65 lamp 1000 lux for 48 hours. As a control sample, the sample exposed to light with a D65 lamp was also stored as a control sample by shading with an aluminum foil, and the stability was evaluated.
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

In SE-HPLC measurement, a TSKgel G3000SWXL column (5 μm; 7.8 mm × 300 mm, manufactured by Tosoh Corporation) is connected to an HPLC system, and a mobile phase with a composition of 50 mmol / L phosphoric acid / 300 mmol / L sodium chloride (pH 6.8) Was flowed at a flow rate of 0.5 mL / min. The concentration of the measurement sample was 10 mg / mL, and was analyzed under the condition of an injection volume of 400 μg. The column temperature was set to 25 ° C., and the wavelength for UV measurement was 280 nm.
The area of the peak detected by SE-HPLC measurement was measured and calculated as a percentage (%) by dividing by the sum of all peak areas including the main peak. The main peak means an active main body peak.
Peaks having a retention time shorter than that of the main peak of SE-HPLC were combined to give a multimer amount, and a peak having a long retention time to give a decomposition product amount. Moreover, when the general term of a multimer and a decomposition product was specified as an impurity, the total of the amount of a multimer and the amount of the decomposition product was made into the amount of impurities. The results are shown in FIGS.
It was shown that the preparation of the present anti-human α9 integrin antibody at around pH 5.0 to 7.0 suppresses the formation of multimers and degradation products.

Example 2 Stabilizing Effect by Selection of Buffer Component
In Table 2, the anti-human α9 integrin antibody obtained in Reference Example is 10 mg / mL, buffer solution (20 mmol / L citric acid (pH 6.0), 20 mmol / L phosphoric acid (pH 6.0), or 20 mmol / L histidine ( The formulation (sample Nos. B-1 to B-3) containing the pH 6.0) is shown. In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH. Each of the formulations diluted with the buffer solution is sterilely filtered with a 0.22 μm filter, filled with 1.3 mL each into glass vials (3 mL capacity), and stoppered with a rubber stopper, and 40 ° C. Exposure was performed for a month and for 48 hours with D65 lamp 1000 lux. As a control sample, the sample exposed to light with a D65 lamp was also stored as a control sample by shading with an aluminum foil, and the stability was evaluated.

The stability (multimeric amount, degraded amount) of anti-human α9 integrin antibody was evaluated. The results are shown in FIGS.
The amount of multimer and the amount of degradation products were analyzed in the same manner as in Example 1 by the SE-HPLC method.
Of the samples stored at 40 ° C. for one month and under D65 lamp irradiation, histidine most suppressed the formation of multimers. Also, all the samples containing citric acid and phosphoric acid were stable.

<< Example 3: Stabilizing effect after long-term exposure by selection of buffer component >>
In Table 3, 10 mg / mL of the anti-human α9 integrin antibody obtained in Reference Example, buffer (20 mmol / L histidine (pH 6.0), 100 mmol / L histidine (pH 6.0), 20 mmol / L citric acid (pH 6.). 0), 20 mmol / L phosphoric acid (pH 6.0), 20 mmol / L succinic acid (pH 6.0), 20 mmol / L 2-morpholinoethanesulfonic acid (hereinafter referred to as MES) (pH 6.0), 20 mmol / L The formulation (sample Nos. B-4 to B-10) containing tartaric acid (pH 6.0) is shown. In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH. The formulation diluted with the buffer solution is sterile filtered with a 0.22 μm filter, filled with 0.5 mL each into glass vials (3 mL capacity), and stoppered with a rubber stopper, D65 lamp 1000 lux · 168 hours An exposure was performed. As a control sample, a sample shielded from light by a method of wrapping in aluminum foil was also stored, and the stability was evaluated.

The stability (multimeric amount) of the anti-human α9 integrin antibody obtained in Reference Example was evaluated. The results are shown in FIG. The amount of multimer was analyzed in the same manner as in Example 1 by the SE-HPLC method.
Of the buffer species tested, histidine most inhibited the formation of multimers. Moreover, the higher the concentration of histidine, the more the generation of multimers was suppressed.

<< Example 4: Stabilizing effect by selection of amino acids, salts, sugars or sugar alcohols>
No additive or arginine (20 mmol / L, 100 mmol / L, or 200 mmol / L) in a formulation containing 10 mg / mL of the anti-human α9 integrin antibody obtained in Reference Example and 20 mmol / L (pH 6.0) of citric acid Sodium chloride (NaCl, 20 mmol / L, 100 mmol / L, or 200 mmol / L), methionine (10 mmol / L, 20 mmol / L, or 100 mmol / L), sorbitol (200 mmol / L), or sucrose (200 mmol / L) Added. In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH. Each of the formulations was sterile filtered with a 0.22 μm filter, then filled with 1.3 mL portions into glass vials (3 mL capacity), stoppered with a rubber stopper, and stored at 40 ° C. for 1 month in the upright state .
In addition, for samples containing arginine, methionine, sodium chloride, sorbitol or sucrose, exposure was performed with a D65 lamp of 1000 lux for 48 hours. In addition, the sample preserve | saved by D65 lamp | ramp also preserve | saved the sample shielded by the method wrapped by aluminum foil as a control | contrast sample, and stability was evaluated.

The amount of multimer was analyzed in the same manner as in Example 1 by the SE-HPLC method.
The results are shown in FIGS.
Arginine, sodium chloride (NaCl), methionine, sorbitol or sucrose examined stabilized or did not affect the stability of the anti-human α9 integrin antibody at 40 ° C. storage conditions.
Arginine, sodium chloride (NaCl), methionine, sorbitol or sucrose also showed a stabilizing effect on light or was stable to light.

Example 5 Stabilizing Effect by Addition of Nonionic Surfactant
In a formulation containing 10 mg / mL of the anti-human α9 integrin antibody obtained in Reference Example, 20 mmol / L (pH 6.0) of histidine, and 140 mmol / L of arginine, polysorbate 80, which is a nonionic surfactant, is 0, 0. It added at 005, 0.01, 0.02, 0.05, or 0.1 w / v%, respectively. In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH. Each of the formulations was sterile filtered with a 0.22 μm filter, then filled into glass vials (10 mL capacity) in 4.4 mL portions, stoppered with a rubber stopper, and physical stability against shaking, freeze-thaw stress was evaluated.
Shaking test, a horizontally placed glass vial was shaken at 150 rpm for 24 hours.
In the freeze-thaw stress test, the sample was stored in a freezer at −80 ° C. in the upright state and frozen, and then the sample was removed from the freezer, stored in the fridge in the refrigerator at 5 ° C., and thawed. The freeze-thaw cycle was repeated three times.

The evaluation results by the light shielding particle counting method are shown in FIG. 15 and FIG.
The light shielding particle counting method was measured using a submerged particle counter (HIAC laboratory type submerged particle counter). The concentration of the measurement sample was 10 mg / mL, and after degassing at 75 Torr and 25 ° C. for 2 hours under standing conditions, analysis was performed under the condition of 0.2 mL injection amount.
The number of insoluble fine particles having a particle diameter of 1.2 μm or more detected by the light shielding particle counting method was measured and calculated as a 1 mL conversion value.
It has been shown that the anti-human α9 integrin antibody suppresses the formation of insoluble microparticles by freeze-thaw and shaking in the nonionic surfactant polysorbate 80-containing preparation.

Example 6 Stability Evaluation Study of a Formulation Containing Histidine, Arginine, Polysorbate 80
After sterile filtration of a formulation containing 10 mg / mL of anti-human α9 integrin antibody obtained in Reference Example, 20 mmol / L of histidine, 140 mmol / L of arginine, and 0.02 w / v% of polysorbate 80 with a 0.22 μm filter, It is filled in a glass vial (10 mL capacity) and sealed with a rubber stopper, shaking, freeze-thaw, light, stability at -20 ° C, 5 ° C and 25 ° C storage by SE-HPLC method, positive It evaluated by the ion exchange chromatography method (CE-HPLC method) and the light shielding particle | grain counting method.
The shaking and freeze-thaw stress tests were conducted in the same manner as in Example 5.
The photostability test was conducted in the same manner as in Example 2.
The stability test at −20 ° C., 5 ° C. and 25 ° C. storage was conducted in the same manner as Example 1.
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH (pH 6.0).

The SE-HPLC method was measured in the same manner as in Example 1, and the light blocking particle counting method was measured in the same manner as in Example 5.
For CE-HPLC measurement, a ProPac WCX-10 column (10 μm; 4 mm × 250 mm, manufactured by Thermo Scientific) was connected to an HPLC system, and mobile phase A: 20 mmol / L MES (2- (N-morpholino) ethanesulfonic acid) pH6. 0, mobile phase B: A mobile phase having a composition of 20 mmol / L MES / 500 mmol / L sodium chloride pH 6.0 was flowed at a flow rate of 0.5 mL / min under the gradient conditions shown in Table 4. The concentration of the measurement sample was 1 mg / mL, and was analyzed under the condition of 10 μg injection volume. The column temperature was set to 40 ° C., and the wavelength of UV measurement was 280 nm.

The area of the peak detected by CE-HPLC measurement was measured and calculated as a percentage (%) by dividing by the sum of all peak areas including peak 1 (main peak). The main peak means the peak of the largest area indicated by the active substance.
Among peaks detected by CE-HPLC, a peak having a large fluctuation during storage was identified and designated as peak 2 and peak 3.
The results are shown in Tables 5 and 6.
The formulation was stable.

Example 7: Evaluation of stability of high concentration liquid preparation containing histidine, arginine, and polysorbate 80
In a formulation containing 100 mg / mL of the anti-human α9 integrin antibody obtained in Reference Example, 20 mmol / L of histidine, 140 mmol / L of arginine, and 0.02 w / v% of polysorbate 80, the pH is 5.0, 5.5, Adjusted to three levels of 6.0. Each of the formulations was sterile filtered with a 0.22 μm filter, and 0.4 mL or 0.3 mL was filled in each micro tube.
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

The stability at 25 ° C. for 1 month storage was evaluated by the SE-HPLC method.
The SE-HPLC method was measured in the same manner as in Example 1.
The results are shown in FIGS. 17-19.
It was stable at any pH.

Example 8 Stabilizing Effect of High Concentration Liquid Preparation by Selection of Amino Acids, Sugar, or Sugar Alcohols
In a formulation containing 100 mg / mL of the anti-human α9 integrin antibody obtained in Reference Example and 20 mmol / L (pH 6.0) of histidine, 20 mmol / L of methionine, 280 mmol / L of sorbitol, 280 mmol / L of sucrose, or arginine The sample to which 140 mmol / L was added was sterile filtered with a 0.22 μm filter, and 0.4 mL or 0.3 mL was filled in each micro tube.
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

The stability at 25 ° C. for 1 month storage was evaluated by the SE-HPLC method.
The SE-HPLC method was measured in the same manner as in Example 1.
The results are shown in FIGS. 20-22.
Formulations containing any of the excipients were also stable.

Example 9 Stabilizing Effect of High Concentration Liquid Preparation by Addition of Nonionic Surfactant
In a formulation containing 100 mg / mL of the anti-human α9 integrin antibody obtained in the reference example, 20 mmol / L (pH 6.0) of histidine, and 140 mmol / L of arginine, polysorbate 80 or poloxamer 188, which is a nonionic surfactant, Each of the formulations to which 0.02 w / v% had been added was sterile filtered with a 0.22 μm filter and then 1.2 mL each was filled into 3 mL glass vials. In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

Physical stability against shaking, freeze-thaw stress was evaluated.
The shaking test and the freeze-thaw stress test were carried out in the same manner as in Example 5.
The evaluation results by the light shielding particle counting method are shown in FIG. 23 and FIG.
The light shielding particle counting method was measured in the same manner as in Example 5.
Both polysorbate 80 and poloxamer 188 inhibited the formation of insoluble fine particles.

Example 10 Evaluation of stability of high concentration liquid preparation containing histidine, arginine, poloxamer 188
Formulated in a formulation containing 100 mg / mL to 200 mg / mL of anti-human α9 integrin antibody obtained in Reference Example, 90 mmol / L (pH 5.8) of histidine, 240 mmol / L of arginine, and 0.02 w / v% of poloxamer 188 Each of the solutions was sterile filtered with a 0.22 μm filter, filled into 1.3 mL portions into 3 mL glass vials, and stoppered with a rubber stopper.
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

The stability upon storage at 5 ° C. and 25 ° C. for 3 months was evaluated by the SE-HPLC method.
The SE-HPLC method was measured in the same manner as in Example 1.
To measure viscosity, connect a capillary (inner diameter: 1.2 mm, made by Anton Paar) to a falling ball viscometer (Automated Micro Viscometer, manufactured by Anton Paar) and allow a metal ball (diameter: 1.0 mm, made by Anton Paar) to fall. It measured by. The capillary temperature was set to 20 ° C., the measurement angle was set to 70 °, and the density was set to 10000 g / cm ³ .
The results are shown in Table 7.
The formulation was stable at any antibody concentration.

Example 11 Evaluation of stability of high concentration liquid preparation containing histidine, arginine, poloxamer 188
Each of the formulations shown in Table 8 containing the anti-human α9 integrin antibody obtained in the reference example was sterile filtered with a 0.22 μm filter, then 1.3 mL each was filled into a 3 mL glass vial and stoppered with a rubber stopper .
In addition, hydrochloric acid and / or sodium hydroxide were added at the time of buffer preparation as needed as a pH regulator so that it might become predetermined pH.

The stability upon storage at 25 ° C. for 3 months was evaluated by the SE-HPLC method and the CE-HPLC method.
Statistical analysis was performed using JMP-11 (manufactured by SAS Institute Inc.).
The SE-HPLC method was measured in the same manner as in Example 1, and the amount of multimer and the amount of degradation products were measured. The CE-HPLC method was measured in the same manner as in Example 6 to measure the amount of main peak.
The results are shown in FIGS. 25-27. The solid line represents the value predicted by statistical analysis based on the obtained data, and the dotted line above and below the solid line represents the width of the value predicted by statistical analysis.
The formulation was stable at an antibody concentration of 200 mg / mL and any histidine concentration and arginine concentration.

According to the present invention, a stable pharmaceutical composition comprising an anti-human α9 integrin antibody, in particular, an anti-human α9 integrin formed by suppressing the formation of a multimer, a degradation product, an insoluble foreign substance and an insoluble microparticle A stable pharmaceutical composition comprising an antibody can be provided.
While the invention has been described in terms of specific embodiments, variations and modifications obvious to one skilled in the art are within the scope of the invention.

The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is the amino acid sequence of the heavy chain of anti-human α9 integrin antibody.
The base sequence represented by SEQ ID NO: 2 is the base sequence of the heavy chain gene of the anti-human α9 integrin antibody.
The amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of the light chain of anti-human α9 integrin antibody.
The base sequence represented by SEQ ID NO: 4 is the base sequence of the light chain gene of the anti-human α9 integrin antibody.

Claims (20)

1. A pharmaceutical composition comprising an anti-human α9 integrin antibody, a pharmaceutically acceptable buffer, and having a pH of 5.0 to 7.0,
   The anti-human α9 integrin antibody is
   (1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A pharmaceutical composition selected from the group consisting of antibodies generated by post-translational modification of
2. The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof. Item 2. The pharmaceutical composition according to Item 1.
3. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutically acceptable buffer is histidine or a pharmaceutically acceptable salt thereof.
4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the pharmaceutically acceptable buffer is 1 mmol / L to 300 mmol / L.
5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising one or more selected from the group consisting of amino acids, salts, sugars and sugar alcohols.
6. Amino acids, salts, sugars or sugar alcohols, such as methionine, arginine, glycine, lysine and ornithine and pharmaceutically acceptable salts thereof, sodium chloride, sucrose, sorbitol, mannitol, trehalose, xylitol, erythritol, The pharmaceutical composition according to claim 5, which is one or more selected from the group consisting of threitol, inositol, dulcitol, arabitol, isomalt, lactitol, and maltitol.
7. The pharmaceutical composition of claim 5 or 6, wherein the amino acids, salts, sugars or sugar alcohols are arginine.
8. The pharmaceutical composition according to any one of claims 5 to 7, wherein the concentration of the amino acids, salts, sugars or sugar alcohols is 1 mmol / L to 400 mmol / L.
9. The pharmaceutical composition according to any one of claims 1 to 8, further comprising a non-ionic surfactant.
10. 10. The pharmaceutical composition according to claim 9, wherein the non-ionic surfactant is polysorbate 80 and / or poloxamer 188.
11. 11. The pharmaceutical composition of claim 9 or 10, wherein the concentration of nonionic surfactant is 0.001% (w / v) to 1% (w / v).
12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the concentration of the anti-human α9 integrin antibody is 1 mg / mL to 1000 mg / mL.
13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the pharmaceutical composition is a liquid formulation or a lyophilized formulation.
14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition is a liquid formulation.
15. A pharmaceutical composition comprising an anti-human α9 integrin antibody, histidine or a pharmaceutically acceptable salt thereof, and having a pH of 5.0 to 7.0, wherein the anti-human α9 integrin antibody is
    (1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A pharmaceutical composition selected from the group consisting of antibodies generated by post-translational modification of
16. The pharmaceutical composition of claim 1 or 15, wherein the post-translational modification of the anti-human α9 integrin antibody is pyroglutamylation of heavy chain N-terminus and lysine deletion of heavy chain C-terminus.
17. Use of a pharmaceutically acceptable buffer, an amino acid, a salt, a sugar or a sugar alcohol as a light stabilizer for the production of a stable pharmaceutical composition containing an anti-human α9 integrin antibody ,
    The anti-human α9 integrin antibody is
    (1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) Use is selected from the group consisting of antibodies generated by posttranslational modification of.
18. The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof. The use according to Item 17.
19. In a pharmaceutical composition containing an anti-human α9 integrin antibody, generation of a degradation product or multimer of the anti-human α9 integrin antibody by exposure to light with a pharmaceutically acceptable buffer, amino acids, salts, sugar or sugar alcohols Is a method of suppressing
    The anti-human α9 integrin antibody is
    (1) An anti-human α9 integrin antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 3, and (2) An anti-human α9 integrin antibody of (1) A method selected from the group consisting of antibodies generated by post-translational modification of
20. The pharmaceutically acceptable buffer is one or more selected from the group consisting of acetic acid, citric acid, phosphoric acid, and histidine, and pharmaceutically acceptable salts thereof. Item 19. The method according to item 19.

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