JPWO2008029908A1 - Stable lyophilized pharmaceutical formulation containing antibody - Google Patents

Abstract

Providing a stable lyophilized pharmaceutical preparation containing an antibody. A lyophilized preparation comprising an antibody and further comprising a crystalline substance and a polyol as an excipient, or the lyophilized preparation wherein the crystalline substance is alanine and the polyol is sucrose or trehalose.

Description

  The present invention relates to the field of antibody pharmaceutical formulations. Specifically, the present invention relates to stable lyophilized pharmaceutical formulations containing antibodies.

  With the progress of biotechnology in recent years, it has become possible to produce a large amount of protein for medical use with high purity using recombinant DNA technology. However, unlike conventional chemically synthesized molecules, proteins have large molecular weights and complicated three-dimensional structures. Therefore, in order to preserve such proteins stably while maintaining stability, special properties are taken into account. Technology is required. Formulations that keep proteins stable need to protect many different functional groups contained in proteins and to maintain higher-order structures that are involved in activity. The development of stable formulations that maintain both activities has become a major challenge.

  An antibody is one of the proteins useful for medical use. In recent years, monoclonal antibodies and polyclonal antibodies have been used in human clinical trials, and pharmaceuticals aimed at treating various diseases such as antitumor, autoimmune diseases and infectious diseases. It is attracting attention in the field. However, purified monoclonal antibodies and polyclonal antibodies are recognized as having the property of easily forming aggregates and granular insoluble fine particles in a solution, and are problematic in the development of antibody preparations. Stabilized antibody formulations that do not have such undesirable properties are desired.

  As one of the methods for stabilizing an antibody, for example, as disclosed in WO98 / 56418, a therapeutically effective antibody that has not been previously lyophilized, an acetate buffer that maintains pH 5.0, and a surfactant. And an aqueous pharmaceutical formulation containing a polyol. Solution formulations are easy and most economical to handle for manufacturers and are easiest to administer to patients. In general, however, solution formulations are subject to chemical degradation (deamidation or oxidation) and physical degradation (aggregation and precipitation), so precise control of storage conditions is necessary to maintain stability. And can be a disadvantage for commercial applications for less stable antibodies.

  In contrast, lyophilization of a product that is relatively unstable in solution can result in a product that is stabilized, and thus has a longer shelf life than a solution formulation. Known in the technical field. (See Non-Patent Document 1). In the dried solid, the decomposition reaction is avoided or remains stable for several years. Therefore, a technique known as lyophilization is often used for injectable pharmaceuticals that exhibit poor stability in aqueous solutions. This step requires Freeze-drying, whereby the ice sublimes from the frozen solution, leaving the original liquid solid dried component. The process involves processing an aqueous solution in a liquid state and filling a dosing container, drying at a low temperature thereby eliminating harmful thermal effects, and storing it in a dried state where it can be more stable. Has a number of advantages to get. In addition, the lyophilized product is usually rapidly soluble and easily reconstituted prior to administration to a patient.

  However, in order to achieve a lyophilized product having the desired characteristics, appropriate care must be taken in selecting conditions and excipients for use in the lyophilization process. Without suitable excipients, most protein formulations are at least partially denatured by freezing and drying, dehydration encountered during lyophilization. In addition, an excipient should be selected that ensures long-term stability with the dried solid.

  The following documents are related to lyophilized preparations containing antibodies.

  Patent Document 1 discloses a lyophilized preparation containing an antibody, a sugar or amino sugar, an amino acid, and a surfactant. However, there is no suggestion that a freeze-dried preparation comprising a combination of alanine and polyol is suitable.

  Patent document 2 is disclosing the lyophilized formulation which consists only of protein, a buffer solution, alanine, and mannitol. However, there is no suggestion that a freeze-dried preparation comprising a combination of alanine and an amorphous polyol is suitable.

  Patent Document 3 discloses a stable lyophilized preparation using 1 to 20 mg of D-mannitol per 1 mg of human monoclonal antibody. However, neither a lyophilized preparation comprising a combination of alanine and an amorphous polyol is suitable, nor does it present a suitable mixing ratio of antibody, alanine and sucrose.

  There is also a lot of scientific literature on the effect of excipients on protein stabilization in protein lyophilized pharmaceutical formulations. However, the theory of the relationship between the structure of a lyophilized product and its stability is not generally accepted. Similarly, the role of polyols and amino acids alone or in combination was not disclosed according to a set of generalized properties, with conflicting results depending on the protein studied and the amount of excipient used.

In addition, the freeze-dried preparations disclosed in the existing patents generate foreign substances, increase the number of polymers, increase the number of oxidants, and so on, and thus cannot find a stable preparation. For example, as disclosed in Patent Document 4 and Patent Document 5, lyophilized preparations containing maltose, which is a reducing sugar, showed an increase in oxidant, particularly during the storage period. As disclosed in Patent Document 2 and Patent Document 3, lyophilized preparations containing mannitol showed an increase in polymer especially during the storage period.
International Publication No. W098 / 22136A2 Pamphlet International Publication No. W097 / 17064 Pamphlet Japanese Patent Laid-Open No. 5-25058 3-504605 International publication WO89 / 11297 pamphlet Remington's Pharmaceutical Sciences, 15th edition, Mack Publishing Co., Easton, Philadelphia, pp 1483-1485

  An object of the present invention is to provide a stable lyophilized pharmaceutical preparation containing an antibody. Specifically, an object is to provide a stable lyophilized pharmaceutical preparation containing a therapeutically effective amount of an antibody and containing a crystalline substance alanine and a non-reducing sugar and non-amorphous polyol.

  The inventors have intensively studied to obtain a stable lyophilized formulation containing a therapeutically effective amount of antibody. As a result, the therapeutically effective amount of the antibody further includes a crystalline substance and a non-reducing sugar and non-amorphous polyol, which occurs during a storage period that is not found in conventionally known lyophilized preparations. It has been found that a freeze-dried preparation having an effect of suppressing the formation of a polymer can be obtained, and the present invention has been completed.

  That is, the present invention is as follows.

[1] A lyophilized preparation containing an antibody and further containing alanine and polyol as excipients.

[2] The lyophilized preparation according to [1], wherein the polyol is a non-reducing sugar and is an amorphous polyol.

[3] The lyophilized preparation according to [1], wherein the polyol is a non-reducing sugar and is an amorphous sugar.

[4] The lyophilized preparation according to [3], wherein the polyol is sucrose or trehalose.

[5] The lyophilized preparation according to any one of [1] to [4], wherein the mass of alanine and polyol with respect to antibody mass 1 is 5/6 to 50.

[6] The freeze-dried preparation according to [5], wherein the polyol mass / alanine mass ratio is 0.5 to 2.5.

[7] The freeze-dried preparation according to [5] or [6], wherein the antibody mass / alanine mass ratio is 0.05 to 3.

[8] The lyophilized preparation according to any one of [1] to [7], wherein the ratio of the mass of the three components of the antibody mass, the alanine mass, and the sucrose mass is 1: 0.3-20: 0.5-30.

[9] The lyophilized preparation according to any one of [1] to [8], further comprising a buffer.

[10] The lyophilized preparation according to [9], wherein the buffer is a glutamate buffer.

[11] The lyophilized preparation according to [9] or [10], wherein the buffer solution has a concentration of 10 mM.

[12] The freeze-dried preparation according to any one of [1] to [11], further comprising a surfactant.

[13] The lyophilized preparation according to [12], wherein the surfactant is polysorbate.

[14] The lyophilized preparation according to any one of [1] to [13], which is a pharmaceutical preparation containing an antibody as an active ingredient.

[15] A method for producing a stable lyophilized preparation comprising adding alanine and polyol to an antibody and lyophilizing the antibody.

[16] The method for producing a freeze-dried preparation of [15], wherein the polyol is a non-reducing sugar and is an amorphous polyol.

[17] The method for producing a lyophilized preparation according to [15], wherein the polyol is a non-reducing sugar and is an amorphous sugar.

[18] The method for producing a freeze-dried preparation according to [16], wherein the polyol is sucrose or trehalose.

[19] The method for producing a freeze-dried preparation according to any one of [15] to [18], wherein the mass of alanine and polyol with respect to antibody mass 1 is 5/6 to 50.

[20] The method for producing a freeze-dried preparation according to [19], wherein the polyol mass / alanine mass ratio is 0.5 to 2.5.

[21] A method for producing a freeze-dried preparation of [19] or [20], wherein the antibody mass / alanine mass ratio is 0.05 to 3.

[22] The method for producing a freeze-dried preparation according to any one of [15] to [21], wherein the ratio of the mass of the three components of the antibody mass, the alanine mass, and the sucrose mass is 1: 0.3 to 20: 0.5 to 30.

  As shown in the Examples, even when the preparation containing the antibody of the present invention is stored at 25 ° C. or 40 ° C. for 3 months, the antibody polymer, degradation product, deamide, and oxidized form increase during the storage period. It was confirmed that the antibody was stable and the biological activity of the antibody was retained. The preparation according to the present invention is stable at least at a low temperature (2 ° C. to 8 ° C.) for 1 year or more, and is stable at least at 25 ° C. for 3 months and / or at 40 ° C. for 1 month.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2006-243144, which is the basis of the priority of the present application.

FIG. 1 is a graph showing changes in the amount of polymer when each lyophilized preparation is stored at 25 ° C. or 40 ° C. The amount of polymer was measured by size exclusion HPLC. Alanine alone indicates that polymer formation is remarkable, but preparations combined with alanine and trehalose or sucrose are stable. FIG. 2 is a graph showing changes in the amount of polymer when each lyophilized preparation is stored at 25 ° C. or 40 ° C. The amount of polymer was measured by size exclusion HPLC. The formulation with R of 0.25 to 2.5 as the ratio of sucrose combined with alanine is stable. FIG. 3 is a graph showing changes in the amount of polymer when each lyophilized preparation is stored at 25 ° C. or 40 ° C. The amount of polymer was measured by size exclusion HPLC. It shows that a preparation having a Q (ratio of antibody mass to alanine mass) of 0.05 to 3 is stable. FIG. 4 shows changes in the amount of polymer when each lyophilized preparation is stored at 25 ° C. or 40 ° C. The amount of polymer was measured by size exclusion HPLC. The anti-HLA-DR antibody and the anti-CD40 antibody show that lyophilized preparations using alanine and sucrose as excipients are stable.

DESCRIPTION OF THE DISCLOSURE RELATED TO THE INVENTION A “stable formulation” is an additive other than an active ingredient that does not contain components that are toxic to the patient to whom the formulation is administered and that does not disrupt as much as possible the patient's biological homeostasis. Is added so that the active ingredient therein retains chemical and / or physical and / or biological stability during storage. “Additives other than active ingredients that do not disrupt patient homeostasis as much as possible” are those that have been confirmed to be sufficiently safe based on past treatment results, or substances that have not been administered in the past. This means that safety is sufficiently predicted by toxicity assessment or other methods. In addition, “maintaining patient homeostasis” means that additives other than the active ingredient have no biological activity unacceptable to the patient and / or isotonic if possible (essentially the same as human blood). It has osmotic pressure).

  Lyophilization is a Freeze drying method often used in the preparation of pharmaceuticals. A liquid composition is prepared and then lyophilized to form a dried cake-like product. This method generally dries an already frozen sample under vacuum to remove the ice, leaving the non-aqueous components in the form of a powder or cake-like material. The lyophilized product can be stored for extended periods of time and at elevated temperatures without loss of biological activity and can be easily reconstituted into a particulate-free solution by adding an appropriate diluent. A suitable diluent is any biologically acceptable liquid in which the lyophilized powder is completely soluble. Water, particularly water for injection, is a suitable diluent because it does not contain salts or other substances that affect antibody stability. The advantage of lyophilization is that the moisture content (which destabilizes the product on long-term storage) can be reduced to a level where various molecular events are greatly reduced. The lyophilized formulation can also easily withstand the physical stresses of transport. The reconstituted product is free of particulates and can therefore be administered parenterally, preferably intravenously or intramuscularly or subcutaneously, without prior filtration.

  An important feature of a lyophilized formulation is the product reconstitution time or the time required to rehydrate. A cake with a highly porous structure is important in order to allow very fast complete rehydration. Cake structure is a function of many parameters including protein concentration, excipient type and concentration, and process parameters of the lyophilization cycle. In general, reconstitution time increases with increasing protein concentration, so short reconstitution time is an important goal in the development of high concentration lyophilized antibody formulations. A long reversion time degrades product quality because the protein is exposed to a more concentrated solution for a longer time. Furthermore, from the user's side, it cannot be administered until the product is completely rehydrated. This is to ensure that the product is free of particulates, the correct dose is administered and its sterility is not affected. That is, rapid rehydration is also convenient for patients and physicians.

  Protein stability is measured by various analytical methods. For example, it is outlined in New Protein Purification Theory and Practice, by RK Scopes, Springer Fairlark Tokyo Publishing. Protein stability includes chemical stability, physical stability and biological stability. “Chemical stability” can be assayed by detecting a chemically altered state of the protein. Chemical changes can be evaluated by size modification such as clipping that can be evaluated by size exclusion chromatography or SDS-PAGE, charge state changes that can be evaluated by ion exchange chromatography (eg, as a result of deamidation), and hydrophobic chromatography Including possible changes in the hydrophilic / hydrophobic state (eg resulting from oxidation) and the like. “Physical stability” includes not producing insoluble particulates and / or turbidity and / or aggregates that can be assessed during visual inspection of color and / or transparency and / or by size exclusion chromatography. “Biological stability” can be evaluated by assaying the binding activity to an antigen that can be evaluated by, for example, size exclusion chromatography or ELISA.

  Proteins useful for therapeutic applications include antibodies. Attempts have been made to use antibodies that bind to proteins expressed on the cell surface and have the effect of inducing cell death or damage to cells for the treatment of cancer and the like. Currently, monoclonal antibodies such as chimeric antibodies (Rituximab) targeting CD20, a receptor on the cell membrane, and humanized antibodies targeting Her2 / neu, are used as target diseases for malignant tumors, and their treatment The effect is recognized. In addition, as disclosed in International Publication No. WO2003 / 033538, a monoclonal antibody (anti-HLA-DR antibody) against an HLA-DR antibody which is a kind of class II major histocompatibility complex (MHC) molecule is also useful. it is conceivable that. An antibody is characterized by a long half-life in blood and a high specificity to an antigen, and is particularly useful as an antitumor agent. For example, in the case of an antibody that targets a tumor-specific antigen, the administered antibody is presumed to accumulate in the tumor, so that it depends on the capture-dependent cytotoxic activity (CDC) and antibody-dependent cytotoxic activity. (ADCC) is expected to attack cancer cells of the immune system. In addition, by binding a drug such as a radionuclide or a cytotoxic substance to the antibody, it becomes possible to efficiently deliver the bound drug to the tumor site, and at the same time, the drug to other non-specific tissues. By reducing the amount reached, side effects can be reduced. When the tumor-specific antigen has an activity that induces cell death, it is possible to administer an antibody having agonistic activity. Also, the accumulation of the tumor-specific antibody and the tumor growth arrest or regression due to the activity of the antibody There is expected. The antibody is considered suitable for application as an antitumor agent due to its characteristics as described above.

  As described above, many antibodies suitable for therapeutic use have been researched, developed and put into practical use, and in the current situation where they are frequently used in the medical field, lyophilization containing more stable antibodies There is a need in the art for formulations.

BEST MODE FOR CARRYING OUT THE INVENTION The “lyophilized preparation” included in this patent is a form in which an active ingredient which is an antibody having a pharmaceutical effect is lyophilized, and the active ingredient is clearly effective in a solid. And does not contain any additional ingredients that may disrupt the homeostasis of the patient to whom the formulation is administered. Here, “assuming that the active ingredient is clearly effective” means that the active ingredient contained therein maintains its activity and does not lose its pharmaceutical effect.

  “Additives other than active ingredients that do not disrupt the patient's biostasis as much as possible” are those that have been confirmed to be sufficiently safe from past treatment results, or substances that have not been administered in the past. This means that safety is sufficiently predicted by toxicity assessment or other methods. That is, the lyophilized pharmaceutical preparation of the present invention may contain an active ingredient which is an antibody having a pharmaceutical effect and other pharmaceutically acceptable additives. In addition, “maintaining patient homeostasis” means that additives other than the active ingredient have no biological activity unacceptable to the patient and / or isotonic if possible (essentially the same as human blood). It has osmotic pressure).

  A “stable formulation” is one in which the active ingredient therein retains chemical and / or physical and / or biological stability upon storage. Preferably it is stable for at least 1 year at least at low temperatures (2 ° C. to 8 ° C.) and preferably stable for at least 3 months at 25 ° C. and / or at least 1 month at 40 ° C. It is stable to vibration. Protein stability is measured by various analytical methods. “Chemical stability” can be assayed by detecting and quantifying the chemically altered state of the protein. Chemical changes include, for example, size modification such as clipping that can be evaluated by size exclusion chromatography or SDS-PAGE, charge changes that can be evaluated by ion exchange chromatography (eg, as a result of deamide), and hydrophilicity / Changes in the hydrophobic state (eg resulting from oxidation) and the like. “Physical stability” includes, for example, the absence of insoluble foreign matter and / or aggregates, and can be evaluated by visual inspection of color and / or transparency and / or size exclusion chromatography. The “biological stability” can be evaluated by examining the binding activity to an antigen that can be evaluated by, for example, a size exclusion chromatograph or ELISA. An antibody retains biological stability when it has not undergone chemical or physical changes. Therefore, when the antibody in the preparation retains chemical stability and physical stability, it can be said that the preparation is stable. That is, whether or not the preparation is stable can be determined by measuring the presence or absence of changes in the chemical and physical properties of the contained antibody. In a stable preparation, antibody polymers, degradation products, deamides, oxidants and the like do not increase so as to reduce the pharmaceutical effect of the preparation during storage, and insoluble foreign matter and turbidity are not observed. The stable formulation of the present invention may be stored at least at a low temperature (2 ° C. to 8 ° C.) for 1 year or more, stored at least 25 ° C. for 3 months, or stored at 40 ° C. for 1 month. The polymer, degradation product, deamide, and oxidant of the antibody do not increase so as to reduce the pharmaceutical effect of the preparation. Moreover, even if the preparation is freeze-thawed or given vibration, the antibody polymer, decomposition product, deamide form, and oxidized form do not increase so as to reduce the pharmaceutical effect of the preparation. The polymer of the antibody in the stable preparation of the present invention does not increase during storage, for example, the polymer for the antibody in the preparation when measured by size exclusion chromatography after being stored at 25 ° C. or 40 ° C. for one month. It is desirable that the ratio of is small. In addition, the degradation product of the antibody in the stable preparation of the present invention does not increase greatly during storage. For example, even if it is stored at 25 ° C. or 40 ° C. for one month, the ratio of the degradation product to the antibody in the formulation is Small is desirable. In addition, the amount of the deamide form of the antibody in the stable preparation of the present invention does not greatly increase during storage. For example, even when stored at 25 ° C. or 40 ° C. for one month, the amount of the deamide form of the antibody in the preparation It is desirable that the ratio is small. Furthermore, the oxidized form of the antibody in the stable preparation of the present invention does not increase greatly during storage. For example, the ratio of the oxidized form to the antibody in the preparation is 1 month even when stored at 25 ° C. or 40 ° C. Small is desirable. The freeze-dried preparation of the present invention is characterized in that the amount of the polymer due to the antibody does not increase during storage. The polymer content and degradation product content can be measured by, for example, size exclusion chromatography, the deamide content can be measured by, for example, ion exchange chromatography, and the oxidant content can be measured, for example, by hydrophobic HPLC.

  The “therapeutically effective amount” of the antibody in the present invention refers to an amount effective for preventing or treating a disease for which the antibody is effective for treatment. A “disease” is any condition that would benefit from treatment with an antibody. This includes chronic and acute diseases or illnesses, including pathological conditions that predispose to mammalian diseases. The therapeutically effective amount of antibody present in the formulation is determined, for example, taking into account the desired dose and mode of administration. About 1 mg / mL to about 200 mg / mL, preferably about 5 mg / mL to about 50 mg / mL, most preferably about 10 mg / mL and / or about 20 mg / mL are exemplary antibody concentrations in the formulation.

  “Antibodies” included in this patent are used in the broadest sense and include in particular monoclonal antibodies, polyclonal antibodies, multispecific antibodies, or antibody fragments so long as they retain the desired biological activity. The antibody included in this patent is not particularly limited as long as it binds to a desired antigen, such as mouse antibody, rat antibody, rabbit antibody, sheep antibody, camel antibody, chicken antibody, chimeric antibody, humanized antibody, human antibody, etc. Can be used as appropriate. The antibody may be a polyclonal antibody or a monoclonal antibody.

  The present invention relates to stable lyophilized pharmaceutical formulations containing antibodies. The antibodies in the formulation are prepared using techniques well known to those skilled in the art that are available in the art to produce antibodies. Exemplary monoclonal antibody preparation methods include: (1) purification of biopolymers used as immunogens and / or preparation of cells overexpressing antigenic proteins on the cell surface, (2) antigens in animal cells Immunization by injection into the blood, and then collecting blood and examining the antibody titer to determine the timing of removal of the spleen and the like, and then preparing antibody-producing cells, (3) myeloma cells (myeloma) Preparation, (4) Cell fusion between antibody-producing cells and myeloma, (5) Selection of hybridoma group producing desired antibody, (6) Division into single cell clones (cloning), (7) In some cases Culture of hybridomas for the production of large quantities of monoclonal antibodies, or rearing of animals transplanted with hybridomas. (8) In some cases, human monoclonal antibodies from antibody-producing cells such as hybridomas A gene encoding a null antibody is cloned, incorporated into an appropriate vector, introduced into a host (eg, mammalian cell line, E. coli, yeast cell, insect cell, plant cell, etc.), and genetic recombination technology is used. Preparation of the recombinant antibody produced, (9) Purification of the antibody thus obtained, (10) Examination of physiological activity and recognition specificity of the monoclonal antibody thus produced, or labeling agent And the like, and the like. Furthermore, polyclonal antibodies can be similarly prepared by methods well known to those skilled in the art.

  The antibody included in this patent includes a heavy chain and / or a light chain having an amino acid sequence in which one or several amino acids are deleted, substituted, or added in each amino acid sequence of the heavy chain and / or light chain of the antibody. An antibody consisting of The partial modification (deletion, substitution, insertion, addition) of amino acids as described above into the amino acid sequence of the antibody of the present invention is introduced by partially modifying the base sequence encoding the amino acid sequence. be able to. This partial modification of the base sequence can be introduced by a conventional method using a known site-specific mutagenesis method. (Proc Natl Acad Sci USA., 1984 Vol81: 5662). The antibodies included in this patent include antibodies having any immunoglobulin class and isotype. The antibody includes a modified antibody in which a subclass is recombined, or a modified antibody in which a heavy chain constant region is further modified. In addition, radionuclides such as iodine, yttrium, indium and technetium (JWGoding, Monoclonal Anibodies: principals and practice, 1993 Academic Press), bacterial toxins such as Pseudomonas aeruginosa toxin, diphtheria toxin, ricin, and methotrexate, mitomycin, caliki. Chemotherapy agents such as amycin (DJKing, Applications and Engineering of Monoclonal Antibodies., 1998 TJ Intenational Ltd .; MLGrossbard., Monoclonal Antibody-Base Therapy of Cancer., 1998 Marcel Dekker Inc), and prodrugs such as Maytansinoid (Chari et al., Cancer Res., 1992 Vol. 52: 127; Liu et al., Proc. Natl. Acad. Sci. USA, 1996 Vol. 93: 8681) Those that further enhance the therapeutic effect are also included.

The antibodies included in this patent also include functional fragments of antibodies. This “functional fragment” means a part (partial fragment) of an antibody that retains at least one action of the antibody on the antigen. Specifically, F (ab ′) ₂ , Fab ′ , Fab, Fv, disulfide bond Fv, single chain Fv (scFv), diabody, and polymers thereof (DJKing., Applications and Engineering of Monoclonal Antibodies., 1998 TJ International Ltd).

  The antibodies included in this patent are polyclonal antibodies and monoclonal antibodies, preferably human antibodies, humanized antibodies or chimeric antibodies. The antibody is preferably IgG, and the IgG subclass is preferably IgG1, IgG2, or IgG4. Further, IgG may be IgG in which amino acid deletion and / or substitution and / or insertion has been carried out by genetic modification in part of the amino acid sequence of the constant region.

  More preferably, HD3, HD4, HD6, HD7, HD8, HD10, HD4G1, HD4G2Ser, HD4G4, HD8G1, HD8G1Ser, HD8G2 are anti-HLA-DR human monoclonal antibodies described in WO2003 / 033538. , HD8G2Ser, HD8G4. Among these, the hybridomas producing HD4, HD6, HD8 and HD10 are FERM BP-7771, FERM BP-7772, FERM BP-7773, and FERM BP-7774, respectively, as an independent administrative agency, industrial technology. It is deposited internationally at the Research Institute's Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan). Further, the hybridomas producing HD3 and HD7 have been deposited internationally at the same center on October 31, 2003 as FERM BP-08534 and FERM BP-08536, respectively. Further, this patent also includes a human monoclonal antibody against CD40, preferably KM281-1-10, KM281-2-10-1-, which are anti-CD40 antagonist antibodies described in WO2002 / 088186. 2, KM283-5, KM225-2-56, KM292-1-24, KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417, F4-465, and KM302, which is an anti-CD40 agonist antibody -1, KM341-1-19, KM643-4-11, 2053, 2105, 3821, 3822, 285, 110, 115, F1-102, F2-103, F5-77, F5-157. Of these, hybridoma clones KM302-1, KM281-1-10, and KM281-2-10-1-2 were dated May 9, 2001 as FERM BP-7578, FERM BP-7579, and FERM BP-7580, respectively. Clone KM341-1-19 and 4D11 were FERMBP-7759 and FERMBP-7758 on September 27, 2001, respectively, and clone 2105 was FERMBP-8024 on April 17, 2002, respectively. It is deposited internationally based on the Budapest Treaty at the Biological Depositary Center (1st East, 1st Street, Tsukuba City, Ibaraki Prefecture, 1st 6th). Furthermore, plasmids having the variable regions of the heavy chain and light chain of F2-103, F5-77 and F5-157 were ATCC PTA-3302 (F2-103 heavy chain) and ATCC PTA-3303, respectively, as of April 19, 2001. (F2-103 light chain), ATCC PTA-3304 (F5-77 heavy chain), ATCC PTA-3305 (F5-77 light chain), ATCC PTC-3306 (F5-157 heavy chain), and ATCC PTA-3307 ( F5-157 light chain), the hybridoma clones F1-102 and F4-465 were dated April 24, 2001 as ATCC PTA-3337 and ATCC PTA-3338, respectively (American Type Culuture Collection, 10801 University Blvd., Manassas, (Virginia, USA) (American National Bacterial Culture Collection, Virginia 20110-2209 Manassas 10801 University Boulevard) has been deposited internationally under the Budapest Treaty. The lyophilized preparation of the present invention is stable when any of the above antibodies is used.

  The lyophilized preparation according to the present invention can be prepared by preparing a solution preparation containing the above antibody and various additives as active ingredients, and then lyophilizing the solution.

  According to a preferred embodiment of the present invention, the lyophilized formulation contains, in addition to the antibody, a polyol, a crystalline substance, and further contains a buffer and a surfactant.

  “Freeze-dried”, “lyophilized” and “freeze-dried” means that the substance to be dried is first frozen and then ice or the freezing solvent is sublimated in a vacuum environment. Means a process to be removed. In order to enhance the stability of the lyophilized product during storage, an excipient may be contained in the preparation prior to lyophilization. The freeze-drying step can be performed by a known method.

  The “additive” included in the present invention includes all components included in the lyophilized preparation other than the active component, such as buffers, pH adjusters, isotonic agents, excipients, stabilizers, surface activity. And preservatives. Moreover, what shows two or more types of effects in a kind of additive component is also contained.

  "Buffer" refers to an additive that moderates changes in pH by the action of acid-base conjugate components. The buffer concentration is about 1 mmol / L to about 50 mmo / L, preferably 5 mmol / L to 20 mmol / L, most preferably about 10 mmol / L, depending on its buffering capacity and / or the desired osmotic pressure. Preferred buffering agents include glutamate (eg, sodium glutamate), citric acid and other organic acid buffers. Most preferred is glutamate.

  A “polyol” is a substance having a plurality of hydroxyl groups and is also used as an excipient in a lyophilized formulation. Contains sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids. Suitable polyols herein have a molecular weight that is less than about 600 KD (eg, in the range of about 120 KD to about 400 KD). A “reducing sugar” is one that can reduce metal ions or contains a hemiacetal group that can react covalently with other amino groups and lysine in a protein. It does not have these properties. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Examples of non-reducing sugars include sucrose, trehalose, sorbose, meletitol, raffinose and the like. Examples of sugar alcohols are mannitol, xylitol, erythritol, threitol, sorbitol, glycerose and the like. The sugar acid includes L-gluconic acid and its metal salt. The use of reducing sugars in lyophilized formulations is not preferred because it promotes the production of oxidants. In addition, when a polyol is used for the purpose of protecting a protein against stress caused by freezing and drying, it is desirable to have an amorphous structure during freezing and drying. Illustrative examples of polyols that have an amorphous structure upon freezing include sucrose, trehalose, sorbitol, and the like. The polyol of the present invention is preferably a non-reducing sugar and an amorphous polyol, or a polyol such as a non-reducing sugar and an amorphous sugar or sugar alcohol. More preferred are non-reducing sugar and amorphous polyols such as sucrose, trehalose, sorbose, meretitol and raffinose, and most preferred are sucrose and trehalose. Moreover, the freeze-dried preparation of the present invention may contain a plurality of polyols.

  The “crystalline substance” only needs to have a crystal structure upon freezing, and includes crystalline amino acids such as alanine and glycine. Particularly preferred is alanine, which is contained in an amount of 5 to 40 mg / mL, preferably 10 to 30 mg / mL, more preferably 15 to 25 mg / mL, and particularly preferably 20 mg / mL.

  Polyols and crystalline materials can act as excipients and tonicity agents. In the present invention, the “excipient” refers to a substance having a function of maintaining a cake shape of a freeze-dried preparation and / or a function of protecting a protein against stress accompanying freezing and drying. In addition to polyols and crystalline materials, a common example used for excipients is lactose. Excipients provide useful properties in that they enhance protein stability during long-term storage. These substances can also act as isotonic agents. An “isotonic agent” refers to one that adjusts the osmotic pressure so that the subject formulation has essentially the same osmotic pressure as human blood. The isotonic preparation preferably has an osmotic pressure of about 250 to 350 mOsm / kg, and the osmotic pressure ratio is preferably about 1 with the osmotic pressure of physiological saline being 1. The alanine used as an excipient in the present invention maintains the cake shape, and the polyol protects the protein against stress associated with freezing and drying.

  In the present invention, a polyol such as sucrose also acts as a stabilizer against antibody aggregation and plays an important role in shortening the time for the lyophilized preparation to be restored to a solution free of particulate matter. The polyol is added to the formulation in an amount that varies depending on the desired osmotic pressure of the formulation. Preferably, the reconstituted lyophilized formulation is isotonic but may be hypertonic or hypotonic.

  “Surfactants” include nonionic surfactants such as polysorbates (eg, polysorbates 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and / or minimizes the formation of particles in the formulation and / or reduces protein adsorption to the container. is there. In the present invention, the surfactant preferably includes polysorbate, and most preferably includes polysorbate 80. The surfactant may be present in the formulation, preferably in an amount of 0.02 mg / mL to 0.1 mg / mL, most preferably about 0.05 mg / mL.

  Examples of pre-lyophilized compositions include about 1 mg / mL to 60 mg / mL IgG antibody, about 10 mmol / L to 20 mmol / L sodium glutamate (pH 5.5), about 0.05 mg / mL polysorbate 80. , And 20 mg / mL alanine and 5 mg / mL to 50 mg / mL sucrose as excipients. The pre-lyophilized formulation is lyophilized to a dry, stable powder that can be easily reconstituted into a particulate-free solution suitable for human administration.

  The present invention provides a stable lyophilized preparation by adding a crystalline substance (most preferably alanine), a non-reducing sugar and an amorphous polyol (most preferably sucrose and trehalose) as excipients to an antibody and freeze-drying it. I will provide a. In essence, the cake structure is maintained in a crystalline phase consisting of a crystalline material and is frozen and dried by an amorphous phase consisting of antibodies and non-reducing sugars and / or amorphous polyols (most preferably sucrose and trehalose). By protecting the protein from stress, a stable lyophilized formulation can be derived.

  The blending ratio and blending amount of alanine and sucrose as excipients used in the present invention are appropriately adjusted according to the antibody concentration as the active ingredient.

  When R is a mass ratio (sucrose mass / alanine mass) or a concentration ratio (sucrose concentration / alanine concentration) of sucrose and alanine present in the lyophilized preparation, R having good stability is 0.25 or more, preferably Was 0.5 to 2.5, more preferably 1 to 2. Most preferably 1.5.

  Freezing containing sucrose / alanine at a ratio of R = 1.5, where Q is the mass ratio of antibody to alanine (antibody mass / alanine mass) or concentration ratio (antibody concentration / alanine concentration) present in the lyophilized preparation The Q with good stability in the dry preparation is 0.05 to 3, preferably 0.05 to 1.5.

  From the combination of R and Q described above, the stable lyophilizing agent comprising antibody, alanine and sucrose has a ratio of the mass of the three components of antibody mass, alanine mass and sucrose mass in the range of 1: 0.3-20: 0.5-30. is there. The ratio of the total mass of alanine and sucrose to the antibody is 1: 5 / 6-50.

  The freeze-dried preparation of the present invention is used after being restored.

  “Restoration” is the time required for “reconstitution time” by rehydrating a lyophilized formulation with a solution to a clarified solution without particles. An important feature of lyophilized formulations is the product reconstitution time or the time required for rehydration, which requires a short time. A cake with a highly porous structure is important in order to allow very fast complete rehydration. Cake structure is a function of many parameters, including protein concentration, excipient type and concentration, and process parameters of the lyophilization cycle. In general, the recovery time increases as the protein concentration increases. Thus, longer reversion times degrade the product quality because the protein is exposed to the more concentrated solution for a longer time. Furthermore, from the user's side, it cannot be administered until the product is completely rehydrated. This is to ensure that the product is free of particulates, the correct dose is administered, and its sterility is not affected by complete rehydration. That is, rapid rehydration is advantageous for patients and doctors.

  Solution formulations can be lyophilized using appropriate drying parameters. The lyophilized formulation retains the stability of the biological activity of the antibody and protects the antibody intended for administration to human subjects from physical and chemical degradation in the final product.

  The lyophilized formulation is rehydrated at the time of use with a diluent (eg, water for injection) to give a particulate-free solution. The reconstituted antibody solution is free of particulate matter after long-term storage of the lyophilized cake at ambient temperature. The reconstituted solution is administered parenterally, preferably intravenously or intramuscularly or subcutaneously to the subject.

  The compositions of the present invention minimize the formation of protein aggregates and particulates in immunoglobulin-containing reagents and ensure that antibodies in solution maintain their immune activity over time. The composition is a sterile pharmaceutically acceptable lyophilized prepared from an aqueous pre-lyophilized formulation comprising antibody, alanine, and polyol and surfactant in a buffer having a neutral or acidic pH. Contains formulation. The composition of the present invention may further contain an excipient other than alanine and polyol and / or an isotonic agent.

  The formulation is administered to mammals, preferably humans, in need of treatment with antibodies, in a known manner, such as intravenous administration as a bolus or by continuous infusion over time, intramuscular, intraperitoneal, intracerebral, subcutaneous, intraarticular. , Intrathecal, intrathecal, oral, topical, or by inhalation route. In a preferred embodiment, the formulation is administered to the mammal by intravenous administration. For such purposes, the formulation is injected, for example, using a syringe or via an infusion line.

  The invention is further illustrated by the following examples. The invention will be fully understood with reference to the following examples. However, these examples do not limit the scope of the invention. Various changes and modifications can be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

Example 1 Combination of Alanine and Various Excipients A freeze-dried preparation was prepared using an antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described in International Publication No. WO2003 / 033538 as an antibody.

In this example, the formulations shown in Table 1 were prepared, and a detailed examination of suitable excipients combined with alanine was conducted.

(1) Preparation of formulation specimen The reagent used in this study was an anti-HLA-DR antibody (approximately 20 mg / mL, according to the method described in the pamphlet of International Publication No. W02003 / 0033538) After July 1, 2007, prepared by Kirin Pharma Co., Ltd., Production Technology Laboratory), sodium L-glutamate monohydrate (Japanese Pharmacopoeia Pharmaceutical Standards), alanine (Ala, Japanese Pharmacopoeia), trehalose (Tor, Japanese Pharmacopoeia), Sucrose (Suc, Japanese Pharmacopoeia), Sodium Chloride (NaCl, Japanese Pharmacopoeia), Hydrochloric Acid (Japanese Pharmacopoeia), Polysorbate 80 (Japanese Pharmacopoeia), Water for Injection (Japanese Pharmacopoeia) Using. Each preparation specimen was prepared by preparing a placebo solution containing no drug substance in advance and replacing the anti-HLA-DR antibody solution with the preparation placebo solution using a NAP column (Pharmacia Biotech). The protein concentration was converted using the OD280nm absorbance coefficient ε = 1.4 and adjusted.

  Each preparation specimen was aseptically filtered using a 0.22 μm filter (Millipore) in a clean bench, and 1 mL each was filled into a 5 mL glass vial (compatible with the Japanese Pharmacopoeia). After filling, half-plugging was performed with a rubber stopper, and lyophilization was performed using a freeze dryer manufactured by Nippon Vacuum Technology Co., Ltd. Further, a solution (placebo) containing no anti-HLA-DR antibody for dilution of the analysis specimen and analysis blank was subjected to aseptic filtration using a 0.22 μm bottle top filter (Nalgen) in a clean bench.

(2) Test conditions In order to evaluate the stability in this example, each preparation specimen was stressed according to the following conditions.

Thermal stability test: Stored in an incubator (manufactured by TABAI ESPEC) controlled at 40 ° C. or 25 ° C. for 1 month and 3 months. Each sample was stored in a low-temperature chamber controlled at 4 ° C until the start of analysis after the heat stress.

(3) Analytical method visual appearance: The shape of the cake was examined with the naked eye under a fluorescent lamp, and an external view was described.

  For the following analysis, after visual inspection, 1 mL of water for injection was added and dissolved again with a syringe and analyzed.

Insoluble foreign matter and turbidity: Under the white light source, the presence of insoluble foreign matter and turbidity was examined with the naked eye at a brightness of about 5000 lux.

Size exclusion HPLC test (SEC): Polymer content and degradation product content were calculated by size exclusion high performance liquid chromatography. The specimen was diluted to 1 mg / mL as necessary, and 20 μL injection was performed at ambient temperature. Separation uses a TSKgel G3000 SWXL 30cm x 7.8mm (Tosoh Corp.) column, 20mmol / L sodium phosphate, 500mmol / L sodium chloride pH7.0 as the mobile phase, and the analysis time is 0.5mL / min. Detection was performed at 215 nm for 30 minutes. In addition, what eluted before a main peak was defined as a polymer, and what eluted after was defined as a decomposition product.

Cation exchange HPLC test (IEC): Deamide content was calculated by cation exchange high performance liquid chromatography. 60 μL of a sample appropriately diluted to 5 mg / mL was injected. Detection was performed at 280 nm using TSKgel BIOAssist S (manufactured by Tosoh Corporation) as a separation column. As mobile phases, 20 mM tartaric acid pH 4.5 for A and 20 mM tartaric acid, 1 M sodium chloride pH 4.5 for B were used, and analysis was performed using optimum gradient conditions. In addition, the deteriorated substance eluted ahead of the main peak was defined as a deamide.

Hydrophobic HPLC assay (HIC): The oxidant content was calculated by hydrophobic chromatography. 20 μL of a sample appropriately diluted to 1 mg / mL using a sample treatment solution (20 mmol / L sodium phosphate, 0.984 mol / L ammonium sulfate, 7.5 mmol / L methionine) was injected. Analysis and detection were performed at 215 nm using TSKgel Butyl-NPR (manufactured by Tosoh Corporation) as a separation column. The mobile phase was 20 mmol / L sodium phosphate, pH 6.5 as A, and 20 mmol / L sodium phosphate, 1.2 mol / L ammonium sulfate, pH 6.5 was used as B. . In addition, the deteriorated substance eluted ahead of the main peak was defined as an oxidant.

Reduced SDS-PAGE assay: The sample solution was diluted to 400 μg / mL as necessary. SDS Sample Buffer (manufactured by Invitrogen), Sample Reducing Agent (manufactured by Invitrogen), and H ₂ O were added to the sample solution and heated at 95 ° C. for 5 minutes to obtain a reduced sample solution. The electrophoresis basket was filled with Tris-Glycine SDS Running buffer (Invitrogen) for electrophoresis. Apply 5 μL of the sample solution to 4-20% Tris-Glycine Gel (Invitrogen) and perform electrophoresis at a constant voltage of about 125 V until the blue color of bromophenol blue contained in the sample solution moves near the bottom of the gel. It was. The gel after electrophoresis was detected by silver staining. In addition, in order to determine the approximate molecular weight of the specimen and the degraded substance, molecular weight markers (including 97 KDa, 66 KDa, 45 KDa, 30 KDa, 20 KDa, and 14 KDa proteins) are run simultaneously.

Non-reducing SDS-PAGE assay: The sample solution was diluted to 400 μg / mL as necessary. LDS Sample Buffer (Invitrogen) and H ₂ O were added to the sample solution to obtain a non-reduced sample solution. The electrophoresis basket was filled with Tris-Acetate SDS Running buffer (manufactured by Invitrogen) for electrophoresis. Apply 5 μL of the sample solution to 3-8% Tris-Acetate Gel (Invitrogen), and perform electrophoresis at a constant voltage of about 125 V until the blue color of bromophenol blue contained in the sample solution moves near the bottom of the gel. It was. The gel after electrophoresis was detected by silver staining. In addition, in order to determine the approximate molecular weight of the specimen and the degraded substance, molecular weight markers (including 220 KDa, 116 KDa, 97.4 KDa, 63.3 KDa, 54.4 KDa, and 36.5 KDa proteins) are run simultaneously.

Osmotic pressure ratio: Osmotic pressure was measured using an automatic osmotic pressure measuring device (OSMO STATION OM-6050 manufactured by ARKRAY, Inc.), and the ratio to the osmotic pressure measured at the same time was calculated. pH: pH was measured using an automatic pH measuring device (such as MP-230 manufactured by METTLER TOLEDO). Measurement was performed after calibration was performed using standard solutions of pH 4, pH 7, and pH 9 at the start of measurement.

Moisture measurement: Moisture content was measured using a trace moisture measuring device (Hiranuma Sangyo AQ-2000) connected to an automatic moisture vaporizer (Hiranuma Sangyo LE-20SA), and the moisture content was calculated.

(4) Results and Discussion FIG. 1 shows changes in the polymer when each lyophilized preparation was stored at 25 ° C and 40 ° C. The amount of polymer was measured by a size exclusion chromatograph. In the preparation using only alanine as an excipient, a polymer increase of about 5% at 40 ° C for 1 month and about 9% at 40 ° C for 3 months was confirmed. In the preparation using alanine and sodium chloride as excipients, foreign substances and turbidity were confirmed by visual inspection with the naked eye after redissolving, and the increase in the polymer was also largely unstable. The preparation combined with trehalose and sucrose was very stable with no increase in polymer even after storage at 40 ° C for 3 months. It was revealed that combining the polyols (trehalose, sucrose) used above with alanine dramatically suppressed the polymer, and it was shown that the stability was high. In addition, in other analysis items (cake shape, foreign matter, turbidity, degradation product, insoluble fine particles, deamide, oxidant), the combination of alanine and polyol shows almost no change before and after heat stress loading, and is stable. there were.

  From this example, it was found that a combination of alanine and polyol was suitable as an excipient of a lyophilized preparation for stabilizing an antibody. Preferably, an amorphous polyol, more preferably a non-reducing and amorphous polyol, and most preferably, trehalose or sucrose is used as the polyol.

Example 2 Sucrose-Alanine Ratio It was found that trehalose or sucrose is most preferred as the polyol combined with alanine in Example 1.

  In this example, sucrose was used as the polyol, the alanine concentration was constant, the sucrose concentration mixed with alanine was changed, and the optimum concentration ratio R for polymer suppression was examined. Here, R represents the sucrose mass / alanine mass present in the lyophilized product.

  A freeze-dried preparation was prepared using an antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described in WO2003 / 033538 pamphlet as an antibody.

In this example, the preparations shown in Table 2 were prepared, and a detailed study of suitable concentrations of sucrose in combination with alanine was conducted.

(1) Materials and Methods The materials and analysis methods used in this example are the same as those shown in Example 1.

(2) Test conditions Similar to the method shown in Example 1, the samples were stored in an incubator controlled by 25 ° C or 40 ° C (manufactured by TABAI ESPEC) for 1 month and 3 months, and a thermal stability test was performed. . Each sample was stored in a low-temperature chamber controlled at 4 ° C until the start of analysis after the heat stress.

(3) Results and Discussion FIG. 2 shows changes in the polymer when each lyophilized preparation was stored at 25 ° C. and 40 ° C. The amount of polymer was measured by a size exclusion chromatograph. It was clarified that the polymer formation was suppressed as the mass of sucrose mixed increased, indicating that the polymer was stable in the range of R = 0.25 to R = 2.5. Furthermore, in the SDS-PAGE analysis, an electrophoretic image showing the same tendency as the result obtained by the size exclusion chromatograph was obtained.

  A cake was formed at any of the concentrations examined in this example, and a clean cake was formed particularly at R = 1.5 or less, showing a shape that can withstand long-term storage. Also, other analysis items (foreign matter, turbidity, insoluble fine particles, deamide, and oxidant) were stable with almost no change observed before and after thermal stress loading.

  In this example, the optimal sucrose mass ratio R (sucrose mass / alanine mass) combined with alanine was clarified as an excipient of the lyophilized preparation for stabilizing the antibody. R having good stability was 0.25 or more, preferably 0.5 to 2.5, more preferably 1 to 2, and further preferably 0.75 to 2. Most preferably 1.5.

Example 3 Examination of influence of antibody concentration A freeze-dried preparation was prepared using an antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described in International Publication No. WO2003 / 033538 as an antibody.

In this example, the preparations shown in Table 3 were prepared, and the effect of the sucrose / alanine formulation (R = 1.5) optimal for stabilizing the antibody obtained in Example 2 on the preparations having different antibody concentrations was evaluated. did. Here, Q was defined as the ratio of antibody mass to alanine mass. That is, Q represents antibody mass / alanine mass in the lyophilized preparation.

(1) Materials and Methods The materials and analysis methods used in this example are the same as those shown in Example 1.

(2) Test conditions In the same manner as in Example 1, the test was stored in an incubator (manufactured by TABAI ESPEC) controlled at 25 ° C or 40 ° C for 1 month and 3 months, and a thermal stability test was performed. . Each sample was stored in a low-temperature chamber controlled at 4 ° C until the start of analysis after the heat stress.

(3) Results and Discussion FIG. 3 shows changes in the polymer when each lyophilized preparation was stored at 25 ° C. and 40 ° C. The amount of polymer was measured by a size exclusion chromatograph. The Q = 3 preparation with the highest antibody concentration (the highest amount of antibody) was about 2% polymer when stored at 40 ° C. for 3 months. In general, it is known that as the antibody concentration increases, the amount of polymer produced increases and becomes unstable. Considering that the amount of polymer in Example 1 antibody concentration 10 mg / mL and alanine alone prescription was about 9% when stored at 40 ° C. for 3 months, polymer formation was sufficiently suppressed. Is clear. In the preparation with Q = 0.05 having the lowest antibody concentration (the least antibody amount), almost no change was observed before and after stress loading, and the preparation was stable. In addition, the other analysis items (foreign matter, turbidity, decomposition product, deamide, and oxidant) were stable with almost no change observed before and after stress loading.

  In this example, it was revealed that a stable lyophilized preparation can be obtained by containing alanine / sucrose at a ratio of R = 1.5 with respect to Q = 0.05 to 3. That is, it shows that a stable lyophilized preparation can be obtained by a lyophilized preparation in which the ratio of the mass of the three components of antibody mass, alanine mass, and sucrose mass is in the range of 1: 0.3 to 20: 0.5 to 30. When expressed as the sum of alanine mass and sucrose mass relative to the antibody mass (antibody mass is 1), it is 1: 5 / 6-50, preferably 1: 1-20, more preferably 1: 1-10, Particularly preferred is 1: 5.

Example 4 Examination with other antibodies In this examination, the optimal alanine / sucrose formulation (R = 1.5) for stabilizing the antibody obtained in Example 2 is published in International Publication No. WO0563981 pamphlet. The stability of the preparation was examined using recombinant fully human IgG4 antibody 4D11 (anti-CD40 antibody) against CD40.

In this example, the preparations shown in Table 4 were prepared, and the effect of antibody type on the stability of the preparation was evaluated.

(1) Materials and Methods The materials and analysis methods used in this example are the same as those shown in Example 1. R = 1.5, Q = 0.5, and the ratio of alanine and sucrose mass to antibody mass was 1: 5.

(2) Test conditions In the same manner as in Example 1, the test was stored in an incubator (manufactured by TABAI ESPEC) controlled at 25 ° C or 40 ° C for 1 month and 3 months, and a thermal stability test was performed. . Each sample was stored in a low-temperature chamber controlled at 4 ° C until the start of analysis after the heat stress.

(3) Results and discussion FIG. 4 shows the change in polymer when each lyophilized preparation was stored at 40 ° C. (Initial ratio: value divided by the amount of polymer before lyophilization. ). The amount of polymer was measured by size exclusion HPLC. Also in the anti-CD40 antibody, suppression of polymer formation was confirmed in the preparation using alanine and sucrose as excipients, and the antibody in this preparation was stably maintained regardless of temperature and time. In addition, in other analysis items (foreign matter, turbidity, decomposition product, deamide, and oxidant), almost no change was observed before and after thermal stress load, and the analysis was stable.

  In this example, it was found that a stable lyophilized preparation containing alanine and sucrose as excipients is stable regardless of the type of antibody.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (22)

  A freeze-dried preparation containing an antibody and further containing alanine and polyol as excipients.   The lyophilized preparation according to claim 1, wherein the polyol is a non-reducing sugar and is an amorphous polyol.   The lyophilized preparation according to claim 1, wherein the polyol is a non-reducing sugar and is an amorphous sugar.   The freeze-dried preparation according to claim 3, wherein the polyol is sucrose or trehalose.   The lyophilized preparation according to any one of claims 1 to 4, wherein the mass of alanine and polyol with respect to antibody mass 1 is 5/6 to 50.   The lyophilized preparation according to claim 5, wherein the polyol mass / alanine mass ratio is 0.5 to 2.5.   The lyophilized preparation according to claim 5 or 6, wherein the antibody mass / alanine mass ratio is 0.05 to 3.   The lyophilized preparation according to any one of claims 1 to 7, wherein the mass ratio of the three components of the antibody mass, the alanine mass and the sucrose mass is 1: 0.3 to 20: 0.5 to 30.   The lyophilized preparation according to any one of claims 1 to 8, further comprising a buffer solution.   The lyophilized preparation according to claim 9, wherein the buffer is a glutamate buffer.   The lyophilized preparation according to claim 9 or 10, wherein the concentration of the buffer solution is 10 mM.   The lyophilized preparation according to any one of claims 1 to 11, further comprising a surfactant.   The lyophilized preparation according to claim 12, wherein the surfactant is polysorbate.   The lyophilized preparation according to any one of claims 1 to 13, which is a pharmaceutical preparation containing an antibody as an active ingredient.   A method for producing a stable lyophilized preparation comprising adding alanine and a polyol to an antibody and lyophilizing the antibody.   The method for producing a lyophilized preparation according to claim 15, wherein the polyol is a non-reducing sugar and is an amorphous polyol.   The method for producing a lyophilized preparation according to claim 15, wherein the polyol is a non-reducing sugar and an amorphous sugar.   The method for producing a lyophilized preparation according to claim 17, wherein the polyol is sucrose or trehalose.   The method for producing a freeze-dried preparation according to any one of claims 15 to 18, wherein the mass of alanine and polyol with respect to antibody mass 1 is 5/6 to 50.   The method for producing a freeze-dried preparation according to claim 19, wherein the polyol mass / alanine mass ratio is 0.5 to 2.5.   The method for producing a lyophilized preparation according to claim 19 or 20, wherein the antibody mass / alanine mass ratio is 0.05 to 3.   The method for producing a freeze-dried preparation according to any one of claims 15 to 21, wherein the ratio of the mass of the three components of the antibody mass, the alanine mass and the sucrose mass is 1: 0.3 to 20: 0.5 to 30.

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