阅读说明：本技术 被封入玻璃容器的冷冻干燥制剂 (Lyophilized preparation sealed in glass container ) 是由 山下正悟 吉泽雄太 于 2019-05-21 设计创作，主要内容包括：本发明提供了一种被封入玻璃容器的、包含治疗剂和表面活性剂的冷冻干燥制剂,其特征在于,该玻璃容器的内表面已被实施硫处理或VIST处理。(The present invention provides a lyophilized preparation comprising a therapeutic agent and a surfactant, which is sealed in a glass container, characterized in that the inner surface of the glass container has been subjected to a sulfur treatment or a VIST treatment.)

1. A lyophilized preparation comprising a therapeutic agent and a surfactant, which is sealed in a glass container, characterized in that the inner surface of the glass container has been subjected to a sulfur treatment or VIST treatment.

2. The formulation of claim 1, wherein the therapeutic agent is a protein.

3. The formulation of claim 2, wherein the protein is an antibody.

4. The formulation of any one of claims 1 to 3, for being redissolved into a solvent such that the concentration of the therapeutic agent in the redissolved solution becomes 0.01mg/mL to 300 mg/mL.

5. The formulation of any one of claims 1 to 4, wherein the surfactant is a non-ionic surfactant.

6. The preparation according to claim 5, wherein the nonionic surfactant is a polyoxyethylene sorbitan fatty acid ester or a polyoxyethylene polyoxypropylene copolymer.

7. The formulation of claim 5 or 6, for being redissolved into a solvent such that the concentration of the non-ionic surfactant in the redissolution becomes 0.001% (w/v) to 1% (w/v).

8. The formulation of any one of claims 1 to 7, which is redissolved such that the redissolution comprises:

0.01mg/mL to 300mg/mL of a therapeutic agent;

0mM to 100mM of a buffer;

0.001% (w/v) to 1% (w/v) of a surfactant; and

1mM to 500mM of a stabilizer and/or 5mM to 500mM of a tonicity modifier.

9. The formulation of any one of claims 1 to 7, wherein the pre-lyophilization solution comprises:

0.01mg/mL to 300mg/mL of a therapeutic agent;

0mM to 100mM of a buffer;

0.001% (w/v) to 1% (w/v) of a surfactant; and

1mM to 500mM of a stabilizer and/or 5mM to 500mM of a tonicity modifier.

10. The formulation of any one of claims 1 to 9, wherein the glass container is a vial or a pre-filled syringe.

11. A kit comprising the formulation of any one of claims 1 to 10 and a solvent for redissolution.

12. A kit comprising a formulation according to any one of claims 1 to 10 and instructions for re-dissolution into a solvent and/or the accompanying text.

13. A method of making a freeze-dried formulation comprising:

(a) providing a glass container having an inner surface of the container that has been subjected to a sulfur treatment or a VIST treatment;

(b) introducing a pre-lyophilization solution comprising a therapeutic agent and a surfactant into the glass container; and

(c) and (5) carrying out freeze drying.

14. A method for preventing or reducing fogging of a glass container of a freeze-dried formulation, comprising:

(a) providing a glass container having an inner surface of the container that has been subjected to a sulfur treatment or a VIST treatment;

(b) introducing a pre-lyophilization solution comprising a therapeutic agent and a surfactant into the glass container; and

(c) and (5) carrying out freeze drying.

15. Use of a glass container, the inner surface of the container of which has been subjected to a sulphur treatment or a VIST treatment, for preventing or reducing fogging of glass during freeze-drying in the manufacture of a freeze-dried formulation.

Technical Field

The present invention relates to a technique for suppressing Fogging of a glass container for a freeze-dried preparation.

Background

Among pharmaceutical preparations, there are many pharmaceutical preparations that use pharmaceutical compositions in liquid form. Liquid formulations must generally be stored at low temperatures and some drugs may deteriorate during storage in liquid form. One possibility to overcome these problems is to freeze-dry the pharmaceutical composition thereof. The pharmaceutical compositions are shipped and stored in dry form and then reconstituted prior to use.

Particularly when the active agent is a protein, the freeze-drying process itself may result in deterioration of the properties of the pharmaceutical composition. In order to prevent the activity of the pharmaceutical composition from being lowered, a surfactant is generally added to the pharmaceutical composition in addition to a cryoprotectant of some kind of sugar or the like.

It is known that the following situations exist: the liquid composition containing the surfactant adheres to a portion above the liquid surface of the inner wall of the vial after filling, and when it is directly freeze-dried, the components of the liquid composition may adhere to a wide range of the inner wall of the vial to remain, thereby giving the vial an "atomized" appearance (patent document 1). That is, the "fogging" area on the inner surface of the glass vial represents: the pre-lyophilized drug solution is moved up to a point beyond the fill level and the pharmaceutical composition is subsequently dried leaving a white residue on the interior surfaces of the vial after the vial has been subjected to the lyophilization process. These residues are not only considered to be significant defects, but also can affect quality checks by visual inspection of the vials or by automated means. Also, poor appearance of the vial may be perceived as problematic by the patient or physician.

Documents of the prior art

Patent document

Patent document 1: japanese Kohyo publication No. 2012-520098

Disclosure of Invention

Problems to be solved by the invention

The present inventors have conducted studies with respect to: a method for suppressing "Fogging" of the inner surface of a container caused by freeze-drying after filling a liquid medicine before freeze-drying into a glass container such as a vial in the preparation of a freeze-dried preparation of a pharmaceutical composition containing a surfactant. The following problems were found in this process: in the method of applying silicone to the inner surface of a glass container disclosed in japanese kokai publication No. 2012-520098, the degree of shrinkage becomes strong in cake formation at the time of freeze-drying, a gap is formed between the wall surface of the container and the cake, and the cake is easily moved. If the cake is easily moved, physical stress at the time of transportation or the like may cause the cake to disintegrate, resulting in degradation of quality. For example, if the cake disintegrates, causing the powder to adhere to the inner wall of the vial, visibility deteriorates, and quality inspection may be affected. Furthermore, even if such cake disintegration is only seen as an apparent defect, it is undesirable, as poor appearance of the vial may be seen as problematic by the patient or physician.

Accordingly, the present invention provides a freeze-dried preparation which prevents fogging of the inner surface of a glass container, further forms a good cake, a method for producing the same, and the like.

Means for solving the problems

The inventors of the present invention have made diligent studies and, as a result, have found that: the present inventors have further studied repeatedly and completed the present invention by performing a sulfur treatment or a VIST treatment on the wall surface (inner surface) of a glass container of a freeze-dried preparation, thereby suppressing the upward movement of a chemical solution on the wall surface of the glass container during freeze-drying, preventing the "fogging" of the wall surface of the glass container after freeze-drying, and realizing good cake formation after freeze-drying.

Namely, the present invention provides the following [1] to [15 ].

[1] A lyophilized preparation comprising a therapeutic agent and a surfactant, which is sealed in a glass container, wherein the inner surface of the glass container has been subjected to a sulfur treatment or a VIST treatment.

[2] The formulation according to [1], wherein the therapeutic agent is a protein.

[3] The preparation according to [2], wherein the protein is an antibody.

[4] The preparation according to any one of [1] to [3], which is to be redissolved into a solvent so that the concentration of the above-mentioned therapeutic agent in the redissolved solution becomes 0.01mg/mL to 300 mg/mL.

[5] The formulation according to any one of [1] to [4], wherein the surfactant is a nonionic surfactant.

[6] The preparation according to [5], wherein the nonionic surfactant is a polyoxyethylene sorbitan fatty acid ester or a polyoxyethylene polyoxypropylene copolymer.

[7] The preparation as recited in [5] or [6], which is to be redissolved in a solvent so that the concentration of the nonionic surfactant in the redissolved solution becomes 0.001% (w/v) to 1% (w/v).

[8] The formulation according to any one of [1] to [7], which is redissolved such that a redissolution comprises:

0.01mg/mL to 300mg/mL of a therapeutic agent;

0mM to 100mM of a buffer;

0.001% (w/v) to 1% (w/v) of a surfactant; and

1mM to 500mM of a stabilizer and/or 5mM to 500mM of a tonicity modifier.

[9] The formulation according to any one of [1] to [7], wherein the solution before freeze-drying comprises:

0.01mg/mL to 300mg/mL of a therapeutic agent;

0mM to 100mM of a buffer;

0.001% (w/v) to 1% (w/v) of a surfactant; and

1mM to 500mM of a stabilizer and/or 5mM to 500mM of a tonicity modifier.

[10] The formulation according to any one of [1] to [9], wherein the glass container is a vial or a prefilled syringe.

[11] A kit comprising the formulation of any one of [1] to [10] and a solvent for re-dissolution.

[12] A kit comprising a formulation according to any one of [1] to [10] and instructions for redissolution in a solvent and/or the accompanying document.

[13] A method of making a freeze-dried formulation comprising:

(a) providing a glass container having an inner surface of the container that has been subjected to a sulfur treatment or a VIST treatment;

(b) introducing a pre-lyophilization solution comprising a therapeutic agent and a surfactant into the glass container; and

(c) and (5) carrying out freeze drying.

[14] A method for preventing or reducing fogging of a glass container of a freeze-dried formulation, comprising:

(a) providing a glass container having an inner surface of the container that has been subjected to a sulfur treatment or a VIST treatment;

(b) introducing a pre-lyophilization solution comprising a therapeutic agent and a surfactant into the glass container; and

(c) and (5) carrying out freeze drying.

[15] Use of a glass container, the inner surface of the container of which has been subjected to a sulphur treatment or a VIST treatment, for preventing or reducing fogging of the glass during freeze-drying in the manufacture of a freeze-dried formulation.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, by suppressing the upward movement of the drug solution on the wall surface of the glass container during freeze-drying, the inner surface of the glass container after freeze-drying can be prevented from being atomized, and the cake can be prevented from being disintegrated, and therefore, a freeze-dried preparation having high quality and excellent appearance can be provided.

Drawings

FIG. 1 shows that in a vial whose surface has been treated with sulfur or VIST, no fogging occurs after freeze-drying.

FIG. 2 shows that shrinkage of the cake was suppressed in the vial whose surface was subjected to sulfur treatment or VIST treatment.

FIG. 3 shows the fogging degree score of freeze-dried products obtained by sealing various chemical solutions in untreated vials and sulfur-treated vials. The sample name indicates [ pharmaceutical active ingredient (API) ID ] - [ concentration ] - [ vial size ].

FIG. 4 shows the fogging degree scores of freeze-dried products obtained by sealing an antibody drug solution in untreated 10mL vials and various surface-treated 10mL vials. The sample name indicates [ pharmaceutical active ingredient (API) ID ] - [ concentration ] - [ vial size ].

Detailed Description

Disclosed is a lyophilized preparation comprising a therapeutic agent and a surfactant, which is sealed in a glass container, wherein the inner surface of the glass container has been subjected to a sulfur treatment or VIST treatment. The freeze-dried preparation of the present invention suppresses fogging of the inner surface of the glass container by suppressing the upward movement of the drug solution on the wall surface of the glass container during freeze-drying, and realizes good cake formation.

In the present invention, "atomization" on the inner surface of the glass container means: the residue of the pharmaceutical composition in the drug solution is attached to the inner wall of the glass container at a position more than the uppermost part of the contact position between the inner wall of the glass container and the cake of the lyophilized preparation. The adhesion of the residue can usually be confirmed by visual observation. It is believed that the fogging of the inner surface of the glass container is caused by: the pharmaceutical composition in liquid form introduced into the glass container is moved up the inner wall and supplied to the freeze-drying in this state. The inhibition of fogging means: the Fogging score (Fogging score) was reduced compared to the case where the glass container before sulfur treatment or before VIST treatment was used. The fogging score is calculated by: the evaluation scores of two parameters (evaluation items), that is, the atomization Area (Area) and the atomization Height (Height), were multiplied.

The atomization degree score is (1) atomizing area score (2) x atomizing height score

(1) The Area of atomization (Area) means: the area of the portion of the wall surface from the uppermost portion of the contact position of the freeze-dried cake in the glass container to the upper end (in the case of a vial, usually a rubber stopper portion) to which the residue of the pharmaceutical composition in the drug solution can adhere. The area of the wall surface from the uppermost part of the contact position of the freeze-dried cake to the upper end of the liquid medicine to which the residue of the pharmaceutical composition can be attached was set to 100%, and the ratio (%) of the nebulized area was calculated, and evaluation scores were recorded according to the following criteria.

(A) When the atomization area was 0%, the evaluation score of the atomization area was set to "0".

(B) When the atomization area is greater than 0% and 5% or less, the evaluation score of the atomization area is set to "1".

(C) When the atomization area was more than 5% and 25% or less, the evaluation score of the atomization area was set to "2".

(D) When the atomization area is more than 25% and 50% or less, the evaluation score of the atomization area is set to "3".

(E) When the atomization area was more than 50% and 75% or less, the evaluation score of the atomization area was set to "4".

(F) When the atomization area is greater than 75% and 100% or less, the evaluation score of the atomization area is set to "5".

(2) The Height of atomization (Height) means the Height (distance) from the uppermost portion of the freeze-dried cake contact position of the inner wall of the glass container to the uppermost (farthest) position where the residue of the pharmaceutical composition in the liquid medicine adheres. The height from the uppermost part of the contact position of the lyophilized cake to the upper end (in the case of a vial, usually a rubber stopper part) to which the residue of the pharmaceutical composition in the drug solution can adhere was set to 100%, and the ratio (%) of the height of atomization was calculated, and the evaluation score was recorded according to the following criteria.

(A) In the case where fogging was not observed, the evaluation score of the fogging height was set to "0".

(B) When the height of atomization is greater than 0% and 5% or less, the evaluation score of the height of atomization is set to "1".

(C) When the height of atomization was more than 5% and 25% or less, the evaluation score of the height of atomization was set to "2".

(D) When the height of atomization was more than 25% and 50% or less, the evaluation score of the height of atomization was set to "3".

(E) When the height of atomization was more than 50% and 75% or less, the evaluation score of the height of atomization was set to "4".

(F) When the height of atomization was greater than 75% and 100% or less, the evaluation score for the height of atomization was set to "5".

When the Fogging degree score is "less than 2", it can be determined that Fogging is not recognized. In one embodiment, the freeze-dried formulation of the invention may have a degree of aerosolization score of less than 2, less than 1.5, less than 1, less than 0.5, or 0.

In the present invention, the glass container is not particularly limited as long as it is a material and a shape suitable for filling and freeze-drying the pharmaceutical composition in a liquid form. As a material of the glass container, for example, borosilicate glass or soda lime glass can be cited, and borosilicate glass is preferable. More specifically, FOR example, materials following the japanese pharmacopoeia (general test method, container/packaging material test method, GLASS container FOR injection test method (3) alkali dissolution test (i) 1 st method), european pharmacopoeia (GLASS CONTAINERS FOR PHARMACEUTICAL USE (GLASS CONTAINERS FOR PHARMACEUTICAL USE)) and united states pharmacopoeia (660. CONTAINERS (CONTAINERS)) can be cited, but are not limited thereto. Further, as the shape of the glass container, there may be mentioned a vial, a prefilled syringe and a cartridge. To facilitate dissolution of the freeze-dried formulation, the pre-filled syringe may be a dual chamber pre-filled syringe. The shape of the cartridge is not particularly limited, for example, a single chamber shape, a double chamber shape, etc., and further, if it is a shape suitable for self-injection, it is more preferable.

In the present invention, the sulfur treatment is usually a chemical treatment for suppressing elution of alkali components from the glass, and is performed by: the eluted components on the glass surface are reacted with an aqueous solution of a sulfur compound such as ammonium sulfate at a high temperature, and removed as water-soluble components. For example, the inner surface of the glass container may be subjected to a sulfur treatment by adding an aqueous ammonium sulfate solution to the container and heating the container at 550 to 650 ℃.

The glass container subjected to the sulfur treatment in the present invention may be a commercially available one, and examples thereof include glass containers provided by murray, salt rice-flour glass co.

In the present invention, the VIST treatment is a treatment for reducing alkali elution from a glass container developed by saikoku corporation, and is carried out by: the inner surface of the glass container is washed with water, an aqueous acid solution, an aqueous surfactant solution, or an aqueous acid solution to which a surfactant has been added. As the acid, organic acids such as formic acid, acetic acid, oxalic acid, phthalic acid, and citric acid, and inorganic acids such as hydrochloric acid, sulfuric acid, and nitric acid can be used, and citric acid is preferably used. The surfactant is not particularly limited, and for example, a nonionic surfactant can be used. VIST treatment can be carried out by known methods, in which case reference can be made to WO 2009/116300.

The glass container subjected to VIST treatment in the present invention may be a commercially available one, and examples thereof include glass containers supplied by Daihu and Special Nitro.

As a treatment for modifying the glass surface, an organosilicon treatment is known in which a glass container is coated with dimethylsiloxane and baked at a high temperature. However, in a glass container treated with silicone, a cake of the freeze-dried preparation is easily shrunk, the cake is easily moved in the glass container, and the cake is easily broken during transportation or the like. In contrast to the above, in a glass container subjected to sulfur treatment or VIST treatment, the cake of the freeze-dried preparation is less likely to shrink, and therefore such a problem does not occur. In particular, the glass container which had been subjected to the sulfur treatment had a high effect of suppressing the shrinkage of the cake. Therefore, in a preferred embodiment of the present invention, a glass container whose inner surface has been subjected to sulfur treatment may be used. Furthermore, the sulfur treatment for glass containers has the following advantages: can be carried out at a lower cost with silicone treatment.

Here, the cake refers to a substance in a state of being freeze-dried after filling a solution before freeze-drying into a glass container.

Here, the occurrence of shrinkage of the cake means a state where the cake is not fixed on the inner surface of the glass container. This state can be easily confirmed by visually observing whether the cake moves within the glass container when the glass container is moved (for example, the glass container is turned upside down).

In the present invention, the surface treatment in the glass container may be performed on the entire inner surface, or may be performed on a part of the inner surface. Preferably, a glass container having an entire inner surface subjected to surface treatment may be used. In one embodiment of the present invention, a glass container having a surface-treated inner surface of the container at a position not lower than the uppermost portion of the contact position of the freeze-dried cake may be used.

The lyophilized formulation of the present invention can be manufactured by lyophilizing a pharmaceutical composition in liquid form (i.e., a pre-lyophilization solution comprising a therapeutic agent and a surfactant) by a method comprising:

(a) providing a glass container having an inner surface of the container subjected to a sulfur treatment or a VIST treatment;

(b) introducing a pre-lyophilization solution comprising a therapeutic agent and a surfactant into the glass container; and

(c) and (5) carrying out freeze drying. Therefore, a method for producing a freeze-dried preparation comprising the above-mentioned steps, and a method for preventing or reducing fogging of a freeze-dried preparation in a glass container comprising the above-mentioned steps are also included in the present invention. According to the above method of the present invention, by suppressing the upward movement of the solution on the wall surface of the glass container before freeze-drying, the fogging of the inner surface of the glass container after freeze-drying is prevented or reduced, and good cake formation is achieved after freeze-drying.

The freeze-drying may be carried out under conditions commonly used in the field of formulation. Freeze-drying is typically carried out in three stages: namely freezing, primary drying and secondary drying.

In the freezing stage, the pharmaceutical composition in liquid form is typically cooled to a temperature below the eutectic point. The solution before lyophilization is usually cooled to-10 ℃ to-80 ℃ under atmospheric pressure (for example, -20 ℃ to-60 ℃), and then frozen.

In the primary drying stage, the pressure is lowered and the temperature is raised in order to sublimate the solvent. The temperature may be from-40 deg.C to 50 deg.C (e.g., -30 deg.C to 40 deg.C). The pressure may be 3Pa to 80Pa (e.g., 5Pa to 60 Pa). A drying stage is typically performed until at least about 90% of the solvent is removed.

In the secondary drying stage, more solvent is removed by raising the temperature. The temperature may be from 10 deg.C to 50 deg.C (e.g., from 20 deg.C to 40 deg.C). The pressure may be 3Pa to 40Pa (e.g., 5Pa to 30 Pa). When the secondary drying stage is complete, the water content of the lyophilisate is usually a maximum of about 5%.

A pre-cooling stage to reduce the temperature to, for example, 2 c to 10 c may be included prior to the freezing stage.

The freeze-dried formulations of the present invention comprise at least one therapeutic agent. Any therapeutic agent that can be lyophilized can be used in the present invention. Therapeutic agents also include proteins, peptides or nucleic acids.

The concentration of the therapeutic agent in the solution prior to lyophilization depends on the type of the therapeutic agent, its use, and the like. The concentration of the therapeutic agent is, for example, in the range of 0.01mg/mL to 300mg/mL, in the range of 0.01mg/mL to 250mg/mL, and in the range of 0.01mg/mL to 200 mg/mL.

In the case where the solution before freeze-drying is a solution having a strong surface-active action, there is a problem that the container is easily atomized during freeze-drying. Alternatively, when the solution before freeze-drying is a solution containing a high concentration of protein, etc., there is a problem that the container is easily atomized during freeze-drying. Thus, in a preferred embodiment, the invention is applied to protein solution formulations, particularly high concentration protein solution formulations for subcutaneous injection and the like. For example, a freeze-dried formulation of the invention may be obtained by: the protein solution preparation was placed in a glass container which had been subjected to sulfur treatment or VIST treatment, and was freeze-dried.

In the present invention, the protein solution preparation is a solution preparation containing a physiologically active protein as an active ingredient. When the solution before lyophilization is a protein solution preparation, the concentration of the protein in the protein solution preparation is, for example, 0.01mg/mL or more, 1mg/mL or more, 10mg/mL or more, 50mg/mL or more, 80mg/mL or more, 100mg/mL or more, 120mg/mL or more, 150mg/mL or more, less than 300mg/mL, less than 250mg/mL, or less than 200 mg/mL. In one embodiment, the concentration of the protein in the solution of the invention before lyophilization is from 0.01mg/mL to 300mg/mL, for example from 1mg/mL to 300mg/mL, from 50mg/mL to 300mg/mL, and the like.

Antibodies are preferred as physiologically active proteins. In a particularly preferred mode, the present invention is applied to an antibody-containing solution preparation containing an antibody. Thus, in a preferred form of the invention, the therapeutic agent is a protein, more preferably an antibody. In a preferred embodiment, the solution before lyophilization in the present invention is a protein solution preparation or an antibody-containing solution preparation, and more preferably a protein solution preparation containing a high concentration of protein or an antibody-containing solution preparation containing an antibody.

In the present invention, the antibody-containing solution preparation means a solution preparation having an antibody concentration of 50mg/mL or more, preferably 80mg/mL or more, more preferably 100mg/mL or more, still more preferably 120mg/mL or more, and yet more preferably 150 mg/mL.

From the viewpoint of production, the upper limit of the antibody concentration in the antibody-containing solution preparation is usually 300mg/mL, preferably 250mg/mL, and more preferably 200 mg/mL. Therefore, the antibody concentration of the antibody solution is preferably 50 mg/mL-300 mg/mL, more preferably 100 mg/mL-300 mg/mL, still more preferably 120 mg/mL-250 mg/mL, and particularly preferably 150 mg/mL-200 mg/mL.

The antibody used in the present invention is not particularly limited as long as it binds to a desired antigen, and may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody in view of stably producing a homogeneous antibody.

The monoclonal antibody used in the present invention includes not only a monoclonal antibody derived from an animal such as a human, a mouse, a rat, a hamster, a rabbit, a sheep, a camel, or a monkey, but also an artificially modified recombinant antibody such as a chimeric antibody, a humanized antibody, or a bispecific antibody. Also included are genetically engineered antibodies in which the variable region and/or constant region of the antibody is artificially altered in order to alter the physical properties of the antibody molecule (specifically, change in isoelectric point (pI), change in affinity for Fc receptors, etc.) for the purpose of improving blood retention and in vivo kinetics.

Furthermore, the immunoglobulin class of the antibody used in the present invention is not particularly limited, and any of IgG such as IgG1, IgG2, IgG3, and IgG4, IgA, IgD, IgE, and IgM may be used, and IgG and IgM are preferred.

Furthermore, the antibody used in the present invention includes not only an antibody having a constant region and a variable region (whole antibody), but also Fv, Fab, F (ab)₂An antibody fragment such as scFv, a single-chain Fv (scFv, sc (Fv)) having a valence of 1 or more and a valence of 2 or more, wherein the variable region of the antibody is bound via a linker such as a peptide linker₂) And diabodies such as scFv dimers, and the like, but the whole antibody is preferable.

The antibody used in the present invention can be prepared by a method well known to those skilled in the art. Basically, hybridomas producing monoclonal antibodies can be produced as follows using known techniques. That is, it can be produced by: desired antigens or cells expressing the desired antigens are used as sensitizing antigens, immunized according to a usual immunization method, the obtained immune cells are fused with known parent cells by a usual cell fusion method, and cells (hybridomas) producing monoclonal antibodies are screened by a conventional screening method. Hybridomas can be prepared, for example, according to the method of Milstein et al (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73: 3-46). When the immunogenicity of the antigen is low, the antigen can be immunized by binding to a macromolecule having immunogenicity such as albumin.

In addition, a recombinant antibody produced as follows can be used: antibody genes are cloned from hybridomas, incorporated into a suitable vector, introduced into a host, and produced as a recombinant antibody using genetic recombination techniques (see, for example, Carl, A.K. Borrebaeck, James, W.Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, published by MACMILLAN PUBLISHERS LTD in the United kingdom in 1990). Specifically, cDNA of the variable region (V region) of the antibody is synthesized from mRNA of the hybridoma using reverse transcriptase. Once the DNA encoding the V region of the antibody of interest is obtained, it is ligated to the DNA encoding the constant region (C region) of the desired antibody and incorporated into an expression vector. Alternatively, the DNA encoding the V region of the antibody may be incorporated into an expression vector comprising the DNA of the C region of the antibody. It is incorporated into an expression vector so that it is expressed under the control of an expression control region such as an enhancer or promoter. The host cell can then be transformed by the expression vector to express the antibody.

In the present invention, a recombinant antibody, such as a Chimeric (Chimeric) antibody or a Humanized (Humanized) antibody, which is artificially altered for the purpose of reducing heterologous antigenicity to humans, can be used. These altered antibodies can be made using known methods. A chimeric antibody is an antibody composed of the variable regions of the heavy and light chains of a non-human mammal such as a mouse antibody and the constant regions of the heavy and light chains of a human antibody, and can be obtained by: the DNA encoding the variable region of the mouse antibody is ligated to the DNA encoding the constant region of the human antibody, incorporated into an expression vector, and introduced into a host to produce the antibody.

Humanized antibodies, also called reshaped human antibodies, are antibodies obtained by grafting Complementarity Determining Regions (CDRs) of a non-human mammal, such as a mouse antibody, into the complementarity determining regions of a human antibody, and general genetic recombination methods thereof are also known. Specifically, from several oligonucleotides prepared to have overlapping portions at the terminal portions, DNA sequences designed to link CDRs of a mouse antibody to Framework Regions (FRs) of a human antibody were synthesized by a PCR method. The DNA obtained is produced by ligating the DNA obtained with a DNA encoding a human antibody constant region, incorporating it into an expression vector, and introducing it into a host (see European patent application laid-open Nos. EP 239400, WO 96/02576). As the FRs of a human antibody to be connected via a CDR, those whose complementarity determining regions form a good antigen-binding site can be selected. Amino acids in the framework regions of antibody variable regions can be substituted, if desired, in order to reshape the complementarity determining regions of human antibodies to form appropriate antigen-binding sites (Sato, K. et al, Cancer Res. (1993)53, 851 856).

As a technique for substituting amino acids of an antibody in order to improve the activity, physical properties, pharmacokinetics, safety, and the like of the antibody, for example, the techniques described below are also known, and the antibody used in the present invention also includes an antibody subjected to the amino acid substitution (including deletion and addition) as described above.

Techniques for performing amino acid substitutions in the variable region of IgG antibodies are reported to be: humanised (Tsurushita N, Hinton PR, Kumar S., Design of humanized antibodies: from anti-Tac to Zenapax., methods.2005 May; 36(1):69-83.) affinity maturation by amino acid substitution of the Complementarity Determining Regions (CDRs) for enhancing binding activity as a starting point (Rajpal A, Beyaz N, berHa L, Cappuccil G, Yee H, Bhatt RR, Takeuchi T, Lerner RA, Crea R., A genetic method for growing the affinity of the affinity by using binding library antibodies, Proc Natl Acad Sci S.2005A.200jun 14; 102-66-8471, S., Takukukura A, R., from Tan anti-Tac to Zenapax, Gec Ha L, Ger Ha R., and B, S.20014, R., G.66-8471, S. Chi, C Natl Acad Sci, S., physicochemical Stability improvement by amino acid substitution of the Framework (FR) (Ewert S, Honegger A, Pluckthun A., Stability improvement of antibiotics for extracellular and intracellular applications: CDR grafting to stable structures and structure-based framework engineering, methods.2004 Oct; 34(2):184-99. Review). In addition, as a technique for performing amino acid substitution in the Fc region of IgG antibodies, a technique for enhancing Antibody-dependent cellular cytotoxicity (ADCC activity) and complement-dependent cellular cytotoxicity (CDC activity) is known (Kim SJ, Park Y, Hong HJ., Antibody engineering for the degradation of therapeutic antibodies, Mol cells.2005Aug 31; 20(1):17-29. Review.). Furthermore, amino acid substitution techniques have been reported for Fc that not only enhance this effector function, but also improve the blood half-life of the antibody (Hinton PR, Xiong JM, Johnfs MG, Tang MT, Keller S, Tsurushita N., An engineered human IgG1 antibody with ringer serum half-life, J Immunol.2006Jan 1; 176(1):346-56., Ghetie V, Popov S, Borvak J, Radu C, Mateso D, Meresan C, Ober RJ, Ward ES, incorasing the serum persistence of IgG An fragment by chromatography, Nat Biotecol.1997; JH; 15: 7: 40). In addition, various amino acid substitution techniques for the constant region for the purpose of improving the physical properties of antibodies are also known (WO 09/41613).

In addition, methods for obtaining human antibodies are also known. For example, a desired human antibody having a binding activity to an antigen can be obtained by sensitizing human lymphocytes with a desired antigen or cells expressing a desired antigen in vitro and fusing the sensitized lymphocytes with human myeloma cells such as U266 (see Japanese examined patent publication (Kokoku) No. 1-59878). In addition, a desired human antibody can be obtained by immunizing a transgenic animal having a complete repertoire of human antibody genes with an antigen (see WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735). In addition, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, phage binding to an antigen can be selected by expressing the variable region of a human antibody as a single chain antibody (scFv) on the surface of the phage by phage display. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. Once the DNA sequence of the scFv that binds to the antigen has been elucidated, an appropriate expression vector containing the sequence can be made to obtain a human antibody. These processes are well known and reference may be made to WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, WO 95/15388. The antibody used in the present invention also includes such human antibodies.

In the case where the antibody gene is once isolated and introduced into a suitable host to produce an antibody, a combination of a suitable host and an expression vector may be used. In the case where eukaryotic cells are used as hosts, animal cells, plant cells, fungal cells may be used. As animal cells, there are known: (1) mammalian cells, such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, (2) amphibian cells, such as Xenopus laevis oocytes, or (3) insect cells, such as sf9, sf21, Tn5, and the like. As the plant cell, a cell derived from the genus Nicotiana (Nicotiana), for example, tobacco (Nicotiana tabacum), is known, and callus culture can be performed thereon. Known fungal cells are yeasts, for example of the genus Saccharomyces, for example Saccharomyces cerevisiae (Saccharomyces cerevisiae), filamentous fungi, for example of the genus Aspergillus (Aspergillus), for example Aspergillus niger (Aspergillus niger). In the case of using prokaryotic cells, there are production systems using bacterial cells. As bacterial cells, escherichia coli (e.coli) and bacillus subtilis are known. The antibody of interest can be obtained by introducing the gene of the antibody of interest into these cells by gene transformation, and culturing the genetically transformed cells in vitro.

Furthermore, the antibody used in the present invention includes antibody modifications. For example, antibodies that bind to various molecules such as polyethylene glycol (PEG) and cytotoxic agents (Farmaco.1999Aug 30; 54(8): 497. su 516., Cancer J.2008. May-Jun; 14(3):154-69.) May also be used. These antibody modifications are also included in the antibody used in the present invention. Such antibody modifications can be obtained by chemically modifying an antibody. These methods have been established in the art.

As the antibody used in the present invention, there can be mentioned an anti-tissue factor antibody, an anti-IL-6 receptor antibody, an anti-IL-6 antibody, an anti-Glypican-3 antibody, an anti-CD 3 antibody, an anti-CD 20 antibody, an anti-GPIIb/IIIa antibody, an anti-TNF antibody, an anti-CD 25 antibody, an anti-EGFR antibody, an anti-Her 2/neu antibody, an anti-RSV antibody, an anti-CD 33 antibody, an anti-CD 52 antibody, an anti-IgE antibody, an anti-CD 11a antibody, an anti-VEGF antibody, an anti-VLA 4 antibody, an anti-HM 1.24 antibody, an anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody), an anti-ganglioside GM3 antibody, an anti-TPO receptor agonist antibody, a blood coagulation factor VIII substitute antibody, an anti-IL 31 receptor antibody, an anti-HLA antibody, an anti-AXL antibody, an anti-CXCR 4 antibody, an anti-NR 10 antibody, a factor IX.

As preferred reshaped humanized antibodies to be used in the present invention, there may be mentioned humanized anti-interleukin 6(IL-6) receptor antibody (see Tolizumab, hPM-1 or MRA, WO92/19759) and humanized anti-HM 1.24 monoclonal antibody. (see WO98/14580), a humanized anti-parathyroid hormone-related peptide antibody (anti-PTHrP antibody) (see WO98/13388), a humanized anti-tissue factor antibody (see WO99/51743), an anti-Glypican-3 humanized IgG1 κ antibody (see codrituzumab, GC33, WO2006/006693), an anti-NR 10 humanized antibody (see WO2009/072604), a bispecific humanized antibody of factor IX and factor X (see ACE910, WO2012/067176), an anti-IL-31 receptor a humanized monoclonal antibody nemolizumab (CIM331) antibody, and the like. As the humanized antibody used in the present invention, particularly preferred are a humanized anti-IL-6 receptor antibody, an anti-NR 10 humanized antibody, a bispecific humanized antibody of factor IX and factor X, and an anti-IL-31 receptor A humanized monoclonal antibody nemolizumab (CIM331) antibody.

As the human IgM antibody, an anti-ganglioside GM3 recombinant human IgM antibody (see WO05/05636) is preferable.

As the low molecular weight antibodies, anti-TPO receptor agonist diabodies (see WO02/33072), anti-CD 47 agonist diabodies (see WO01/66737), and the like are preferable.

In the present invention, an antibody having a low isoelectric point (low pI antibody) is an antibody having a low isoelectric point, which is difficult to exist in nature. Examples of the isoelectric point of such an antibody include, but are not limited to, 3.0 to 8.0, preferably 5.0 to 7.5, more preferably 5.0 to 7.0, and particularly preferably 5.0 to 6.5. It should be noted that natural (or normal) antibodies are generally considered to have isoelectric points in the range of 7.5-9.5.

Further, as the antibody used in the present invention, it is preferable to change the pI of the antibody by changing the amino acid residues exposed to the surface of the antibody to lower the pI of the antibody. Such an altered pI antibody is an antibody having a pI that is 1 or more, preferably 2 or more, more preferably 3 or more lower than that of the antibody before alteration. Examples of such an antibody having an altered pI include, but are not limited to, SA237(MAb1, H chain/SEQ ID NO: 1, L chain/SEQ ID NO: 2) which is an anti-IL-6 receptor antibody described in WO2009/041621, a humanized antibody which is an anti-NR 10 human, and a fully humanized NS22 antibody produced by the method described in example 12 of WO 2009/072604.

Examples of the amino acid residue exposed on the surface of the antibody in the case of the H chain variable region include, but are not limited to, amino acid residues selected from the group consisting of H, H82, H105, H108, H110, and H112, which are amino acid residues numbered by Kabat. In the case of the L chain variable region, amino acid residues selected from the group consisting of L1, L3, L7, L8, L9, L11, L12, L16, L17, L18, L20, L22, L24, L27, L38, L39, L41, L42, L43, L45, L46, L49, L53, L54, L55, L57, L60, L63, L65, L66, L68, L69, L70, L74, L76, L77, L79, L80, L81, L85, L100, L103, L105, L106, and L107, which are amino acid residues based on Kabat numbering, may be mentioned, but not limited thereto.

In the present invention, "alteration" refers to substitution of an original amino acid residue with another amino acid residue, deletion of an original amino acid residue, addition of a new amino acid residue, and the like, and preferably refers to substitution of an original amino acid residue with another amino acid residue.

It is known that there are charged amino acids among amino acids. Generally, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids). As the negatively charged amino acid (negatively charged amino acid), aspartic acid (D), glutamic acid (E), and the like are known. The other amino acids are referred to as amino acids having no charge.

In the present invention, the amino acid residue after the alteration is preferably selected as appropriate from the amino acid residues contained in any one of the following groups (a) or (b), but is not particularly limited to these amino acids.

(a) Glutamic acid (E), aspartic acid (D)

(b) Lysine (K), arginine (R), histidine (H)

Further, in the case where the amino acid residue before the change already has a charge, changing the amino acid residue to an amino acid residue having no charge is one of preferred embodiments.

That is, the changes in the present invention include (1) substitution of an amino acid having a charge with an amino acid having no charge, (2) substitution of an amino acid having a charge with an amino acid having a charge opposite to that of the amino acid, and 3) substitution of an amino acid having no charge with an amino acid having a charge.

The isoelectric point value can be measured by isoelectric point electrophoresis, which is known to those skilled in the art. In addition, the theoretical isoelectric point values can be calculated using gene and amino acid sequence analysis software (Genetyx et al).

Antibodies with altered charges of amino acid residues can be obtained by: altering a nucleic acid encoding the antibody, culturing the nucleic acid in a host cell, and purifying the antibody from the host cell culture. In the present invention, "altering a nucleic acid" refers to altering a nucleic acid sequence to form a codon corresponding to an amino acid residue introduced by the alteration. More specifically, it refers to a codon in which the nucleotide sequence of the nucleic acid is changed so that the codon forming the amino acid residue before the change is introduced by the change. That is, the codon encoding the amino acid residue to be changed is replaced with the codon encoding the amino acid residue introduced by the change. Such nucleic acid modification can be appropriately performed by using techniques known to those skilled in the art, for example, site-specific mutagenesis, PCR mutagenesis, and the like.

The protein used in the present invention may be a physiologically active protein other than an antibody, which can be used as a drug. Examples of such physiologically active proteins include, but are not limited to, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), Erythropoietin (EPO), hematopoietic factors such as thrombopoietin, interferons, cytokines such as IL-1 and IL-6, monoclonal antibodies, Tissue Plasminogen Activator (TPA), urokinase, serum albumin, blood coagulation factor VIII, leptin, insulin, stem cell growth factor (SCF), and the like.

The physiologically active protein has substantially the same biological activity as that of a physiologically active protein of a mammal, particularly a human being, and includes a protein of natural origin and a protein obtained by a gene recombination method, but is preferably a protein obtained by a gene recombination method. The physiologically active protein produced by the gene recombination method may use a protein obtained as follows: bacteria such as Escherichia coli; a yeast; proteins obtained by producing cultured animal-derived cells such as Chinese Hamster Ovary (CHO) cells, C127 cells, and COS cells, and extracting, isolating, and purifying the cells by various methods. The protein obtained by the gene recombination method includes a protein having an amino acid sequence identical to that of a natural protein, or a protein having one or more of the above amino acid sequences deleted, substituted or added and having the above biological activity. The physiologically active protein also includes a protein chemically modified with PEG or the like.

Examples of the physiologically active protein include proteins having sugar chains. The source of the sugar chain is not particularly limited, and a sugar chain added to mammalian cells is preferred. Among mammalian cells, for example, CHO cells, BHK cells, COS cells, human-derived cells, and the like are present, and among them, CHO cells are most preferable.

For example, in the case where the physiologically active protein is G-CSF, any G-CSF purified at high purity can be used as the G-CSF. The G-CSF in the present invention can be produced by any method, and it is possible to use G-CSF obtained by: culturing a cell line of human tumor cells, G-CSF obtained by various methods of extraction, isolation and purification therefrom, or genetically engineering bacteria such as escherichia coli; yeast; G-CSF is produced from cultured cells of animal origin such as Chinese Hamster Ovary (CHO) cells, C127 cells, and COS cells by various methods including extraction, isolation, and purification. Preferably, the recombinant DNA is produced by Escherichia coli, yeast or CHO cells using a genetic recombination method. Most preferably, G-CSF is produced using CHO cells using a gene recombination method. Further, G-CSF chemically modified by PEG or the like is also included (see International patent application publication No. WO 90/12874).

The buffer used in the protein-containing solution preparation is prepared by using a buffer, which is a substance for maintaining the pH of the solution. In the antibody-containing solution preparation, the pH of the solution is preferably 4 to 8, more preferably 5.0 to 7.5, still more preferably 5.5 to 7.2, and still more preferably 6.0 to 6.5. The buffer usable in the present invention is a pharmaceutically acceptable buffer capable of adjusting the pH in the above range. Such buffers are known to those skilled in the art of solution formulation, and for example, inorganic salts such as phosphate (sodium or potassium salt), sodium bicarbonate, and the like; organic acid salts such as citrate (sodium or potassium salt), sodium acetate, sodium succinate and the like; or acids such as phosphoric acid, carbonic acid, citric acid, succinic acid, malic acid and gluconic acid. In addition, a buffer such as Tris and MES, MOPS, HEEPS (Good's buffer), histidine (e.g., histidine hydrochloride), glycine, etc. may be used.

In the antibody-containing solution preparation, the buffer is preferably a histidine buffer or a glycine buffer, and particularly preferably a histidine buffer. The concentration of the buffer is usually 1 mM-500 mM, preferably 5 mM-100 mM, more preferably 10 mM-20 mM. In the case of using a histidine buffer, the buffer preferably contains 5 mM-25 mM histidine, more preferably 10 mM-20 mM histidine.

The antibody-containing solution preparation is preferably stabilized by adding a stabilizer for an antibody suitable as an active ingredient. For "stable" antibody-containing solution formulations, no significant change is observed for at least 12 months, preferably 2 years, more preferably 3 years at refrigeration temperatures (2-8 ℃); or no significant change is observed at room temperature (22-28 ℃) for at least 3 months, preferably 6 months, more preferably 1 year. For example, the total amount of the dimer and the decomposition product is 5.0% or less, preferably 2% or less, and more preferably 1.5% or less after storage at 5 ℃ for 2 years, or the total amount of the dimer and the decomposition product is 5.0% or less, preferably 2% or less, and more preferably 1.5% or less after storage at 25 ℃ for 6 months.

The freeze-dried formulation of the present invention comprises at least one surfactant.

Typical examples of the surfactant include nonionic surfactants such as sorbitan fatty acid esters such as sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate; glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyristate, and glycerin monostearate; polyglyceryl fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, and decaglyceryl monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, and polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol tetrastearate and polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glycerin monostearate; polyethylene glycol fatty acid esters such as polyethylene glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hardened castor oil such as polyoxyethylene castor oil and polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides having an HLB of 6 to 18, such as polyoxyethylene fatty acid amides, such as polyoxyethylene stearic acid amide; anionic surfactants such as alkyl sulfates having an alkyl group with 10 to 18 carbon atoms, for example, sodium cetyl sulfate, sodium lauryl sulfate, and sodium oleyl sulfate; polyoxyethylene alkyl ether sulfates such as sodium polyoxyethylene lauryl sulfate having an average number of moles of ethylene oxide added of 2 to 4 and an alkyl group having 10 to 18 carbon atoms; alkyl sulfosuccinate salts having an alkyl group with 8 to 18 carbon atoms, such as sodium lauryl sulfosuccinate; natural-based surfactants such as lecithin, glycerophospholipids; sphingomyelin such as sphingomyelin; sucrose fatty acid esters of fatty acids having 12 to 18 carbon atoms. The freeze-dried preparation of the present invention may contain one or a combination of two or more of these surfactants.

Preferred surfactants are nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters and polyoxyethylene polyoxypropylene copolymers, with polysorbate 20, 21, 40, 60, 65, 80, 81, 85 and pluronic type surfactants being particularly preferred, and polysorbate 20, 80 and pluronic F-68 (Poloxamer 188) being most preferred.

The amount of the surfactant added to the solution before lyophilization is usually 0.0001% (w/v) to 10% (w/v). In one embodiment, the concentration of the nonionic surfactant in the solution before lyophilization of the present invention is from 0.001% (w/v) to 1% (w/v), for example from 0.001(w/v) to 5% (w/v), and from 0.005(w/v) to 3% (w/v).

The freeze-dried preparation of the present invention may suitably contain pharmaceutically acceptable component ingredients as necessary. For example, suspending agents, solubilizers, preservatives, anti-adsorbents, diluents, excipients, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, and the like can be mentioned.

Examples of the suspending agent include methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

Examples of the solubilizer include polyoxyethylene hardened castor oil, polysorbate 80, niacinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, castor oil fatty acid ethyl ester, and the like.

Examples of the tonicity agent include sodium chloride, potassium chloride, calcium chloride, and the like.

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

Examples of the anti-adsorption agent include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, polyoxyethylene hardened castor oil, polyethylene glycol, and the like.

Examples of the sulfur-containing reducing agent include reducing agents having a mercapto group such as N-acetyl cysteine, N-acetyl homocysteine, lipoic acid, thiodiglycol, monoethanolamine thioalcohol, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.

Examples of the antioxidant include chelating agents such as erythorbic acid (erythorbic acid), dibutylhydroxytoluene, butylhydroxyanisole, α -tocopherol, tocopheryl acetate, L-ascorbic acid and salts thereof, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate or disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.

Further, the freeze-dried preparation of the present invention may suitably contain a stabilizer and/or a tonicity modifier as necessary.

In the present specification, a stabilizer refers to a pharmaceutically acceptable additive that protects a therapeutic agent and/or formulation from chemical and/or physical degradation during manufacture, storage, and administration. The chemical and physical decomposition pathways of drugs are reviewed in Cleland et al (1993), crit. Rev. Therr. drug Carrier Syst.10(4):307-77, Wang (1999) int.J.Pharm.185(2):129-88, Wang (2000) int.J.Pharm.203(1-2):1-60 and Chi et al (2003) Pharm.Res.20(9): 1325-36. Examples of the stabilizer include sugars, amino acids, polyols, cyclodextrins (e.g., hydroxypropyl- β -cyclodextrin, sulfobutylethyl- β -cyclodextrin and β -cyclodextrin), polyethylene glycols (e.g., PEG3000, PEG3350, PEG4000 and PEG6000), albumins (e.g., Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA)), salts (e.g., sodium chloride, magnesium chloride and calcium chloride) and chelating agents (e.g., EDTA). The stabilizer may be present in the pre-lyophilization solution or the reconstitution solution in an amount of about 1mM to about 500mM, preferably in an amount of about 10mM to about 300mM, and more preferably in an amount of about 100mM to about 300 mM.

In the present specification, tonicity adjusting agent means a pharmaceutically acceptable additive for adjusting the tonicity (osmotic pressure property) of a solution before lyophilization or a redissolved solution. Examples of tonicity adjusting agents include sodium chloride, potassium chloride, glycerin, and any of amino acids or sugars. The tonicity modifier may be present in the solution prior to lyophilization or in the redissolution in an amount of about 5mM to about 500mM, preferably in an amount of about 50mM to about 300 mM.

In a preferred form, the pre-lyophilization solution of the present invention comprises:

about 0.01mg/mL to about 200mg/mL of a therapeutic agent;

0mM to about 100mM of a buffering agent;

from about 0.001% (w/v) to about 1% (w/v) of a surfactant; and

about 1mM to about 500mM of a stabilizer and/or about 5mM to about 500mM of a tonicity modifier.

In a preferred embodiment, where the therapeutic agent is a protein, the pre-lyophilization solution of the present invention comprises:

about 50mg/mL to about 300mg/mL of protein;

0mM to about 100mM of a buffering agent;

from about 0.001% (w/v) to about 1% (w/v) of a surfactant; and

about 1mM to about 500mM of a stabilizer and/or about 5mM to about 500mM of a tonicity modifier.

Before use, the lyophilized preparation of the present invention is dissolved in a pharmaceutically acceptable solvent such as water for injection and administered to a subject in the form of a re-dissolved solution. The composition of the redissolved solution may be the same as or different from the composition of the solution prior to lyophilization.

The concentration of the therapeutic agent in the redissolution depends on the type of the therapeutic agent, the use thereof, and the like. The concentration of the therapeutic agent is, for example, in the range of 0.01mg/mL to 300mg/mL, in the range of 0.01mg/mL to 250mg/mL, and in the range of 0.01mg/mL to 200 mg/mL.

In one embodiment, when the therapeutic agent of the present invention is a protein, the lyophilized preparation of the present invention is redissolved in a solvent so that the concentration of the protein in the redissolved solution is, for example, 0.01mg or more, 1mg/mL or more, 10mg/mL or more, 50mg/mL or more, 80mg/mL or more, 100mg/mL or more, 120mg/mL or more, 150mg/mL or more, less than 300mg/mL, less than 250mg/mL, or less than 200 mg/mL. In one embodiment, the lyophilized preparation of the present invention is redissolved in a solvent such that the protein concentration in the redissolved solution is 0.01mg/mL to 300mg/mL, for example, 1mg/mL to 300mg/mL, 50mg/mL to 300mg/mL, or the like.

In one embodiment, the lyophilized preparation of the present invention is resuspended in a solvent so that the concentration of the nonionic surfactant in the redissolved solution is from 0.001% (w/v) to 1% (w/v), for example from 0.001% (w/v) to 5% (w/v), and from 0.005% (w/v) to 3% (w/v).

In a preferred mode, the freeze-dried preparation of the present invention is redissolved such that the redissolution comprises:

about 0.01mg/mL to about 200mg/mL of a therapeutic agent;

0mM to about 100mM of a buffering agent;

from about 0.001% (w/v) to about 1% (w/v) of a surfactant; and

about 1mM to about 500mM of a stabilizer and/or about 5mM to about 500mM of a tonicity modifier.

In a preferred mode, in the case where the therapeutic agent is a protein, the freeze-dried preparation of the present invention is redissolved such that the redissolution comprises:

about 50mg/mL to about 300mg/mL of protein;

0mM to about 100mM of a buffering agent;

from about 0.001% (w/v) to about 1% (w/v) of a surfactant; and

about 1mM to about 500mM of a stabilizer and/or about 5mM to about 500mM of a tonicity modifier.

The osmotic pressure ratio of the redissolved solution prepared from the freeze-dried formulation of the present invention is about 0.5 to 4, more preferably about 0.7 to 2, and even more preferably about 1.

The freeze-dried preparation of the present invention is dissolved in a pharmaceutically acceptable solvent and then administered by subcutaneous injection, intravenous injection, intramuscular injection, etc. With respect to a preparation for subcutaneous injection, in the case where the amount per administration is large (for example, in the case where the therapeutic agent is an antibody, about 80mg to 200 mg), there is a limitation in the amount of an injection solution. The lyophilized preparation of the present invention can prepare a re-dissolved solution containing a therapeutic agent at a high concentration, and is therefore particularly suitable for subcutaneous injection.

In the present invention, when a vial is used, the capacity of the vial is 2mL to 100mL, for example, 3mL to 20 mL.

In one embodiment, the amount of solution prior to lyophilization is from 10% to 90%, such as from 20% to 80% of the vial capacity. For example, when the vial has a capacity of 10mL, the amount of the solution before lyophilization is 1mL to 9mL, for example, 2mL to 8 mL.

In one embodiment, the amount of the drug solution after redissolution is 10% to 90%, for example 20% to 80%, of the capacity of the vial. For example, when the vial has a capacity of 10mL, the amount of the drug solution after redissolution is 1mL to 9mL, for example, 2mL to 8 mL.

All prior art documents cited in this specification are hereby incorporated by reference.

The present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not limited to these.

Examples

Example 1: effect of suppressing fogging of glass vials by Sulfur treatment or VIST treatment

The degree of fogging of the inner surface of the glass container when a liquid medicine containing a protein was placed in a glass vial which had been subjected to sulfur treatment, VIST treatment, silicone treatment or no treatment and freeze-dried was evaluated.

The following were used for sample bottles: 10mL of a white lot of vials (material: borosilicate glass; untreated; manufactured by tradename Rice-Mill Co., Ltd.); the vials were a 10-mL white sulfur batch (material: borosilicate glass; sulfur-treated; manufactured by Rex Kogyo Co., Ltd.), a 10-mL VIST vial (material: borosilicate glass; subjected to VIST treatment; manufactured by Katsumadai Kaisho Co., Ltd.) and a 10-mL TopLyo (registered trademark) vial (material: borosilicate glass; subjected to silicone treatment; manufactured by SCHOTT AG). The vials were dry heat sterilized at 250 ℃ for 120 minutes and then used.

As protein-containing drug solutions, solutions of 30mg/mL of antibody (CIM331), 6mmol/L of Tris buffer, 75mmol/L of sucrose, 45mmol/L of arginine and 1880.15 mg/mL of poloxamer were prepared. CIM331 is an anti-human IL-31RA antibody described in WO 2010/064697 a1 and WO 2016/167263 a1, and is prepared by methods known to those skilled in the art according to the descriptions in the above patent documents.

5mL of a liquid medicine containing a protein was put into a vial, frozen at-45 ℃ using Triomaster manufactured by Council vacuum Co., Ltd, and then subjected to primary drying at a temperature close to the disintegration temperature under vacuum conditions, and then subjected to secondary drying at 30 ℃ under vacuum conditions.

After freeze-drying, the extent of surface Fogging of the glass container was evaluated by Fogging score. The fogging degree score was calculated by multiplying two parameters (evaluation items), namely, evaluation scores of the fogging Area (Area) and the fogging Height (Height) (see table 1 below).

The atomization degree score is (1) atomizing area score (2) x atomizing height score

(1) The Area of atomization (Area) means: the area of the portion of the glass wall surface from the uppermost portion of the contact position of the lyophilized cake to the rubber stopper portion where the residue of the pharmaceutical composition in the drug solution adheres. The area from the uppermost part of the contact position of the freeze-dried cake to the glass wall surface of the plug part of the rubber plug was set to 100%, and the ratio (%) of the atomization area was calculated, and the evaluation score was recorded according to the following criteria.

(A) When the atomization area was 0%, the evaluation score of the atomization area was set to "0".

(B) When the atomization area is greater than 0% and 5% or less, the evaluation score of the atomization area is set to "1".

(C) When the atomization area was more than 5% and 25% or less, the evaluation score of the atomization area was set to "2".

(D) When the atomization area is more than 25% and 50% or less, the evaluation score of the atomization area is set to "3".

(E) When the atomization area was more than 50% and 75% or less, the evaluation score of the atomization area was set to "4".

(F) When the atomization area is greater than 75% and 100% or less, the evaluation score of the atomization area is set to "5".

(2) The Height of atomization (Height) means the Height (distance) from the uppermost portion of the contact position of the freeze-dried cake to the uppermost (farthest) position where the residue of the pharmaceutical composition in the liquid medicine adheres. The height from the uppermost part of the contact position of the freeze-dried cake to the plug part of the rubber plug was set to 100%, and the percentage of the atomization height (%) was calculated, and the evaluation score was recorded according to the following criteria.

(A) In the case where fogging was not observed, the evaluation score of the fogging height was set to "0".

(B) When the height of atomization is greater than 0% and 5% or less, the evaluation score of the height of atomization is set to "1".

(C) When the height of atomization was more than 5% and 25% or less, the evaluation score of the height of atomization was set to "2".

(D) When the height of atomization was more than 25% and 50% or less, the evaluation score of the height of atomization was set to "3".

(E) When the height of atomization was more than 50% and 75% or less, the evaluation score of the height of atomization was set to "4".

(F) When the height of atomization was greater than 75% and 100% or less, the evaluation score for the height of atomization was set to "5".

If the Fogging score (Fogging score) "is less than 2", it can be determined that Fogging is not recognized.

[ Table 1]

Degree of atomization score [ area ] x [ height ]

The results are shown in FIG. 1. The atomization scores for the sulfur-treated vial, VIST-treated vial and silicone-treated vial were 0, and no atomization was confirmed. On the other hand, the untreated vial had a degree of fogging score of 16, and the presence of fogging was confirmed. The following possibilities are believed to exist: these treatments reduce the roughness of the glass vial surface, thereby reducing wettability and making fogging difficult to occur.

Example 2 no cake shrinkage occurred in sulfur-treated vials and VIST-treated vials

For the vials subjected to various surface treatments, freeze-dried preparations were prepared in the same manner as in example 1, and the state of cake was evaluated.

The results are shown in FIG. 2. In the silicone-treated vials, it was found that the cake shrank upon freeze-drying, forming a void between the container wall and the cake, allowing the cake to move easily within the vial. On the other hand, in the sulfur-treated vial and the VIST-treated vial, the cake did not shrink and good cake formation was achieved (data not shown). If the tortillas are easily moved, physical stress during transportation or the like may cause the tortillas to disintegrate, resulting in a decrease in quality. Thus, it has been demonstrated that sulfur-treated vials and VIST-treated vials inhibited fogging upon freeze-drying and formed a good cake, and thus were superior to silicone-treated vials.

Example 3 evaluation of degree of atomization of freeze-dried products obtained by enclosing various chemical solutions in various surface-treated vials Score and pie migration ratio

< method >

Test samples: protein solutions as described in table 2 were prepared.

[ Table 2]

Vial: the vials shown in table 3 were used in the same manner as in example 1. However, for both untreated vials (normal) and sulfur treated vials (sulfur), 3mL vials were also used. The vials were dry heat sterilized at 250 ℃ for 120 minutes and then used.

[ Table 3]

The evaluation method comprises the following steps: about 3mL of the test sample was placed in a 10mL vial, and about 1mL of the test sample was placed in a 3mL vial. After freezing at-45 deg.C, primary drying was carried out at a temperature close to the disintegration temperature under vacuum conditions using Triomaster II-A04 (manufactured by Kyowa vacuum Co., Ltd.), and secondary drying was carried out at 30 deg.C under vacuum conditions. Then, the fogging suppression effect of the glass vial was evaluated in the same manner as in example 1. Further, the movement ratio of the cake was calculated by observing the movement of the cake when the vial was inverted, and the shrinkage ratio of the cake (detachment from the vial) was evaluated.

Cake migration ratio (%). number of samples moved ÷ number of observed samples × 100

< results >

Effect of suppressing fogging of glass vial

The results are shown in table 4 and fig. 3 and 4.

[ Table 4]

As shown in table 4 and fig. 3, in the sulfur-treated vials, the fogging score was less than 2 for all test samples, and no fogging was observed. On the other hand, in the untreated vial, the degree of atomization scores were 2 or more for all the test samples, and atomization was observed.

As shown in table 4 and fig. 4, the degree of atomization scores of the freeze-dried products obtained by sealing the respective antibody solutions in the vial subjected to VIST treatment and the vial subjected to silicone treatment were less than 2, and no atomization was observed.

Percentage of cake shrinkage (tables 4 and 5)

The results are shown in tables 4 and 5.

[ Table 5]

Table 5: cake transfer ratio when inverting lyophilized products obtained by enclosing antibody liquid medicine in untreated 10mL vials and various surface-treated 10mL vials

In the silicone-treated vials, cake movement occurred in all vials of all test samples (cake movement ratio 100%). On the other hand, the VIST-treated vial showed a lower rate of cake migration in all test samples than the silicone-treated vial, and the sulfur-treated vial showed a lower rate of cake migration. Thus, the sulfur-treated vial and the VIST-treated vial showed less cake shrinkage than the silicone-treated vial.

Industrial applicability

According to the present invention, there can be provided: a product having excellent appearance and quality due to prevention of fogging and cake disintegration of the inner surface of a glass container of a freeze-dried formulation. In addition, by preventing fogging of the inner surface of the glass container, false detection by the automatic checker is reduced and the quality control process of the freeze-dried formulation is made more efficient.