TW200826974A - Stable lyophilized pharmaceutical preparation comprising antibody - Google Patents

Abstract

Disclosed is a stable, lyophilized pharmaceutical preparation comprising an antibody. Specifically disclosed is a lyophilized preparation comprising an antibody and further comprising a crystalline substance and a polyol as excipients, wherein the crystalline substance may be alanine and the polyol may be sucrose or trehalose.

Description

200826974 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the field of pharmaceutical preparations of antibodies. In particular, the present invention relates to a freeze-dried pharmaceutical preparation which is stable with an antibody. [Prior Art] ^In recent years, the development of biological jade sciences has used the recombinant DNA technology to produce a large amount of medical proteins with excellent purity. However, unlike the previous synthetic molecules, the molecular weight of the protein is large and its three-dimensional structure is complex. Therefore, in order to keep such a protein in a stable state, it is necessary to consider it. Special technology for physical properties. To maintain a stable protein preparation, it is necessary to protect the many different Luyue b-based 'X' contained in the protein must maintain the multi-dimensional structure related to activity; in the field of protein medicine, develop and maintain the stability and activity of the protein. The formulation of the stability of the person is becoming a big issue. As one of the proteins useful in the medical field, there are antibodies. In recent years, Q monoclonal antibodies or multiple anti-systems have been put into clinical trials for the purpose of anti-tumor or (7) autoimmune diseases and infectious diseases. The pharmaceutical industry is receiving attention from the industry. However, the purified monoclonal antibody or a plurality of antibodies have been found to have a property of forming agglomerates and granular insoluble microparticles and granules in a solution, which is a problem in the development of antibody preparations. It is expected that a stabilized antibody preparation that does not have such undesirable properties will be desired. As one of the methods for stabilizing an antibody, for example, in the case of International Publication No. W098/56418, there is disclosed an aqueous pharmaceutical preparation containing an antibody which has been previously freeze-dried and is therapeutically effective, and has a value of 124402. .doc 200826974 Acetate buffer, surfactant, and polyol in 5.0. Solution formulations are easy to handle and most economical for the manufacturer, and are most easily administered to patients. However, in general, if it is a solution preparation, it is affected by chemical decomposition (deamination reaction or oxidation reaction) and physical deterioration (aggregation and precipitation). Therefore, in order to maintain stability, it is necessary to properly control storage conditions. For antibodies with low stability, it will become a disadvantage in their commercial applications. In view of this, it is known in the art that a relatively unstable product in a solution is freeze-dried, and it is possible to obtain a stable product, thereby having a longer storage time and stability than a solution preparation. (Refer to Non-Patent Document 1). In the dried solids, the decomposition reaction can be avoided, or it can be kept stable for several years. Therefore, as a known technique for freeze-drying, it is often applied to an injectable pharmaceutical which exhibits poor stability in an aqueous solution. This process must be freeze-dried (Freeze_drying), whereby the ice is subcooled; the solid solution in the east is sublimed and the solids of the residual liquid are passed through the components of (4). In this step, the aqueous solution is processed in a liquid state and filled in, and dried, at a low temperature, to eliminate harmful heat effects, and then it can be preserved in a more stable dry state. In addition, the lyophilized product is usually rapidly dissolved and can be easily reconstituted before administration to a patient. However, in order to produce a cold, east dried product having the desired characteristics, it is necessary to pay attention to the conditions and excipients used in the cold roll drying step. Without proper excipients, most of the protein preparations will be denatured at least by the cold rolling and drying and dehydration encountered during cold drying. In addition, sexual excipients. % Select to ensure the long-term stability of the dried solid. For the freeze-dried antibody containing the antibody, the I agent has the following literature. Patent Document 1 discloses that -Tan Tan 3 has a frozen earth preparation of an antibody, an anthracene or an amino group, an amino acid, and a surfactant. However, it does not suggest that a lyophilized preparation containing human, sigma, and sigma is suitable. Patent Document 2 discloses that the cold medicinal saponin is composed of only protein, a buffer solution, propylamine, and mannitol. However, it does not suggest that a freeze-dried preparation comprising a combination of alanine and amorphous polysaccharides is known. Patent Document 3 discloses that a sandalwood 1 mg human monoclonal antibody is used as a lyophilized preparation of 疋~(9) mg of D-mannitol. However, it has not been suggested that a lyophilized preparation comprising a combination of alanine and amorphous sulphate is suitable as a 'and' does not suggest a better mixing ratio of antibody, alanine or sulphur. In the freeze-dried pharmaceutical preparation of protein shell, there is a large amount of scientific literature on the effect of the agent on the stabilization of the protein > % 〇 疋. However, the theory of the relationship between the structure of the cold-dried product, the re-recovery, the re-/, and the relationship between the female and the female, is usually not accepted. Similarly, the use of _, the use of sputum alcohol and amino acid or the combination of these is not disclosed according to the general nature of the group, depending on the protein being studied and the excipients used. The quantity 'discovered has the opposite result. Further, in the cold-dried preparation disclosed in the prior patent, there are cases in which (4) production, polymer increase, and oxidized body increase occur, and the preparation of the ampoule is not found, such as Patent Document 4 or Patent Document 5. $, in the cold; East dry preparation containing as a reducing sugar maltose, especially during the storage period 124402.doc 200826974 increased oxidant. As shown in Patent Document 2 or Patent Document 3, the freeze-dried preparation containing mannitol is particularly increased by 0 Ο during storage. Patent Document 1: International Publication No. WO098/22136A2 Patent Document 2: International Publication No. Patent Document No. WO97/17064, Japanese Patent Laid-Open No. Hei 5-25058, Patent Document 4, Japanese Patent Application Laid-Open No. Hei No. 3-504605 Patent Document 5: Non-Patent Document 1 of International Publication No. WO 89/11297 Remington's Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., Easton, Philadelphia, pp. 1483-1485 [Invention] The problem to be solved by the present invention is to provide a freeze-dried pharmaceutical preparation which is stable with an antibody. Specifically, it is an object of the present invention to provide a stable freeze-dried pharmaceutical preparation comprising a therapeutically effective amount of an antibody, a proline which is a crystalline substance, and a non-reducing sugar and an amorphous polyol. The technical means for solving the problem, the inventors have made efforts to obtain a stable and effective preparation of a therapeutically effective amount of the antibody; As a result of the study, it has been found that a therapeutically effective amount of the antibody further contains a crystalline substance, and is a non-returnable sugar and an amorphous guest agent. The present invention has finally been completed by a cold-dried preparation which does not have a fruit which is produced in the sputum and which inhibits the fruit produced during storage. Chengxin 124402.doc 200826974 That is, the present invention is as follows. [π kinds of freeze-dried preparations, [2] The lyophilized preparation according to [1], wherein the polyol is a non-reducing sugar and is an amorphous polyol. [] [1] A lyophilized formulation wherein the polyol is a non-reducing sugar and is an amorphous sugar.

冷冻 [] [3] a lyophilized preparation, wherein the polyol is a cold-dried dry preparation of sucrose or trehalose 0 [5]^[1] to [4], wherein, relative to the antibody Berry 1, The mass of alanine and polyol is 5/6~50. The freeze-dried preparation of [][] wherein the ratio of the mass of the polyol to the mass of the alanine is from 0.5 to 2.5. [] Cold as in [5] or [6]; East dry preparation in which the ratio of antibody mass/alanine mass is 0.05 to 3. ' [8] As in any of [1] to [7], the quality of alanine and the quality of sucrose [9] such as [1] to [8]. The lyophilized preparation of the invention wherein the mass ratio of the antibody mass to the component 3 is 1:0.3 to 20:0.5 to 30. a lyophilized preparation, which further comprises a lyophilized preparation according to [10], [9], wherein the buffer is a face acid buffer in which the concentration of the buffer is 10, such as m or (10), cold 涞 dry _ mM 〇[12], such as any one of [1] to [11], 7 freeze-dried preparation, which further contains a boundary 124402.doc 200826974 surfactant agent is polysorbate = 3] such as the cold rape (10) preparation, ^ A pharmaceutical preparation containing the antibody Π4] such as the cold μ of any one of (1) to [13] as an active ingredient. It includes ···································································· The method is a method in which the polyol is non-Η7], such as "Μ, the manufacture of a cold-drying preparation, a reducing sugar, and an amorphous sugar. [18] ;东干_@ ^ Sugar or trehalose. ' ', medium polyol is sugar cane [19] such as [15] to [18], which is relative to the quality of the antibody, lyophilized preparation method 5/6~50. The mass of the bis-acid and the polyol is the mass of the alcohol / [2〇] such as U9] The ratio of the mass of the cold-rolled dry preparation alaline is 0.5~2.5. The method wherein the antibody [21] such as [19] or [20] has a mass/alanine mass ratio of 〇〇5\3 [22], wherein any one of [15] to [21], wherein the antibody The quality and the propylamine g-management, the method of cold drying the preparation, the mass ratio of the three components, the inner and the sucrose quality | is 1:0.3~20: 〇·5~30 〇 the effect of the invention 124402.doc 200826974 & clinic The antibody-containing preparation of the present application, even if kept at 25 C or 40 C for 3 months, does not increase the polymer, decomposition product, deamamine, and oxidant of the antibody during storage. For stability, and 'the antibody is also confirmed to remain biologically active. The preparation of the present application, at least at low temperature (2 ° C to 8. Under the armpit for more than 1 year, and at least 3 at 25 C) The month and/or the stability of one month at 4 rc. This specification contains the contents described in the specification and/or drawings of the patent application No. 2006-243 144, which is the priority of the present application. [Embodiment] The description of the disclosure relating to the present invention, "a stable preparation", does not include a knife which is toxic to a patient who is administered the preparation, and adds activity which does not interfere with the physiological constantity of the patient as much as possible. Additions other than the ingredients, and the active ingredients therein are kept chemically and/or physically and/or biologically stable during storage. The so-called "additions other than the active ingredients that do not interfere with the physiological constants of the person as much as possible" ", according to the past Among the substances that have been confirmed to have sufficient safety, or those that have not actually been used in the past, can be fully predicted by the toxicity of cells or animals, or by other methods. Keeping the physiological constants of the patient, including: additives other than the active ingredient do not have biological activity that is not acceptable to the patient, and/or isotonic if possible (having essentially the same osmotic pressure as human blood) Lyophilization is a freeze-drying method often used in the preparation of pharmaceuticals. A liquid composition is prepared, which is then lyophilized to form a dried cake. This method, generally and ancient, 124402.doc -12- 200826974 糸: The sample which has been cold-blown is dried and removed in a vacuum; the water is directly residual (4) aqueous component in the form of a powder or a cake-like substance. The dried product can be stored for a long time at high temperature without losing its biological activity, and can be easily restored to a solution containing no granules by adding a suitable diluent: Suitable diluent, biological Any liquid that is tolerated and is cold; a liquid in which the East dry powder can be completely dissolved. Water, especially water for injection, is a suitable diluent because it does not contain a salt or other substance f which affects the stability of the antibody. Cold; the advantage of East Drying is that the moisture content (the moisture will become unstable during long-term storage) is reduced to the extent that the sub-events are greatly reduced. Further, the freeze-dried preparation is easily resistant to the physical pressure of the transport. The reconstituted product does not contain granules, so that it can be administered orally (4) without oral administration, or (4) by intravenous or intramuscular or subcutaneous administration. An important feature of the cold-bundled formulation is the recovery time of the article or the time necessary for rehydration. In order to be rehydrated very quickly and completely, what is important is a cake having a highly porous structure. The cake structure is a function of many parameters including protein concentration, type and concentration of excipients, and process parameters for the cold drying cycle. In general, if the protein concentration rises, the recovery time increases, so a short recovery time is an important target in the development of high-concentration freeze-dried antibody preparations. The longer the recovery time, the better the quality of the product is caused by the protein being exposed to the further concentrated solution for a longer period of time. Further, from the user's point of view, the product cannot be administered until the product is completely rehydrated. The aim is to ensure that the product is free of granules, is administered correctly, and that its sterility is not affected. 124402.doc -13- 200826974 ie Rapid rehydration is beneficial to both patients and doctors. The stability of the protein is determined by various analytical methods. For example, it is outlined in "The Theory and Practice of New Protein Refinement" (RK Sc〇pes, Beijing Publishing). Protein stability includes chemical stability, physical stability, and biological stability. "Chemical stability. Sex" can be identified by detecting the chemical state of a protein. Chemical changes include, for example, size modifications such as size exclusion chromatography or ζ-s 仃 evaluation, or changes in charge states that can be evaluated by ion exchange chromatography (for example, results of dedeamine production) and Hydrophobic chromatography can be used to evaluate changes in the hydrophilic/hydrophobic state (for example, the result of oxidation). "Physical stability" includes visual inspection using color and/or transparency and/or evaluation by size exclusion chromatography without insoluble microparticles and/or turbidity and/or agglomeration. The "biological stability" can be evaluated, for example, by identifying the antigen-binding activity which can be evaluated by size exclusion chromatography or ELISA. Proteins useful for therapeutic use may comprise antibodies. The manufacturer is attempting to combine an antibody with a protein expressed on the surface of a cell, and an antibody having an action of inducing cell death or injury to a cell is used in the treatment of cancer or the like. At present, a monoclonal antibody such as a chimeric antibody (Rituximab) targeting the receptor which is present on the cell membrane, and a human antibody targeting Her2/neu is used as a target disease, and it can be seen. treatment effect. Further, as disclosed in the case of International Publication No. WO〇2〇〇3/〇33538, it can be considered as HLA- as a π-type main tissue affinity gene complex 124402.doc •14-200826974 (MHC) molecule. A monoclonal antibody (anti-HLa_dr antibody) of the DR antibody is also effective. The antibody has a long half-life in blood and a high specificity to an antigen, and is particularly effective as an antitumor agent. For example, if an antibody targeting a tumor-specific antigen is used, it is presumed that the administered antibody will accumulate in the tumor 2, and thus the immune system may be expected to be complement-dependent cytotoxic activity (CDC) or antibody-dependent cytotoxic activity. (ADCC) attacks cancer cells. It is also expected that by combining a drug p such as a radioactive nucleus or a cytotoxic substance with an antibody, the bound drug can be efficiently delivered to the tumor site while reducing the amount of the agent reaching the non-specific tissue. Reduce side effects. Further, in the case of having an activity for inducing cell death by a tumor-specific antigen, it is expected that aggregation of a tumor-specific antibody, and tumor growth or atrophy due to antibody activity can be expected by administering an antibody having agonistic activity. . As described above, an antibody can be considered to be useful as an antitumor agent depending on its characteristics. As described above, a large number of antibodies suitable for therapeutic use have been studied, opened, and put into practical use, and are frequently used in medical facilities. In this case, a freeze-dried preparation containing an antibody having more stability is considered as This area has become a necessity. The so-called "freeze-dried preparation" contained in this patent is a form in which the active ingredient, which is a pharmaceutical effect, is freeze-dried, and is in a form in which the active ingredient is made into a solid effect, and does not contain Other components that can interfere with the physiological constants of the patient to which the formulation is administered. Here, the phrase "the active ingredient is made to have a clear effect" means that the active ingredient contained therein maintains its activity without losing the effect. 124402.doc -15- 200826974 The so-called "additional ingredients other than the active ingredients that do not interfere with the physiology of the patient as much as possible" means that the person has confirmed that there is sufficient safety according to the actual treatment in the past, or in the past Among the substances that have no actual results, the safety of cells and animals can be fully predicted by other methods. #, The cold-dried pharmaceutical preparation of the present invention may comprise: an active ingredient as a medicinal antibody, and other additives permitted by the medicinal means. Also, 'the so-called "maintaining the physiology of the patient" includes: the additive other than the active ingredient does not have biological activity that is not acceptable to the patient, and/or isotonic if possible (having essentially the same as human blood) Osmotic pressure) and the like. By "stable preparation" is meant one in which the active ingredient remains chemically and/or physically and/or biologically stable during storage. Preferably, it is at least low temperature (2. 〇 to 8. 〇 under the 丨 under the stability of the leap year, and, more preferably, at 25 ° C for at least 3 months and / or at 4 〇 t at least Keep _ month: stability, the formulation of cold reduction, light irradiation and vibration is stable. The female virginity of the protein is determined by various analytical methods. "Chemical stability, the state of chemical change by quantitative detection of protein For the purpose of identification, "chemical changes" include, for example, size modification such as size exclusion chromatography or shear evaluation, and charge change that can be evaluated by ion exchange chromatography (for example, the result of producing deguanamine) And the use of a hydrophobic layer: a change in the hydrophilic/hydrophobic state (for example, oxidation: result), etc. The "physical stability" includes the color and/or transparency of the object. Size exclusion chromatography can be used to evaluate the situation of non-productive, non-micron particles and/or turbidity and/or agglomerates. Then, "Life::: 124402.doc -16- 200826974 J can be used, for example, by Borrowing The small exclusion chromatography or the binding activity of EUSA and the like to the antigen is evaluated and evaluated. When the body undergoes chemical or physical changes, the antibody retains biological ampoule. Therefore, in the preparation The antibody in the chemical stability and physical stability: the 'form time' can be considered to be stable in the formulation. That is, whether the preparation is An sigma by measuring whether the antibody contained in the sputum produces chemical and physical properties changes. In the preparation, the polymer of the antibody, the decomposition product, the deamidamine, and the emulsifier are equal to the extent that the effect of the preparation is not increased until the preparation is reduced, and no insoluble foreign matter or turbidity is found. The stability of the present invention! y agent, even if it is stored at least at low temperature (2t to 8 〇 under the armpit for more than i years, or until =; C for 3 months, or even at 40 ° C for 1 month, antibody polymerized knives, The removal of the amine and the oxidant will not increase to the extent that the effect of U is lowered. In addition, even if the preparation is subjected to cold beam melting or vibration, the polymer, decomposition product, deacetylamine or oxidant of the antibody is not -i曰 added to P cattle low preparation effect To the extent that the polymer of the antibody in the stable preparation of the present invention does not increase in storage, for example, at 25 ° C or (10). After storage for 1 month at C, it is better to measure by size exclusion chromatography. The ratio of the polymer to the antibody in the preparation is small. X, the antibody decomposition product in the stable preparation of the present invention does not increase significantly during storage, for example, for 1 month after adding or listening, preferably The ratio of the decomposition product to the antibody in the preparation is small. Further, the amount of the deamidamine of the antibody in the stable preparation of the present invention is not greatly increased during storage, for example, at 40 C for one month. 'It is preferred that the ratio of the valeramine to the antibody in the preparation is small and the oxygen of the antibody in the stable preparation of the present invention is 124402.doc -17-200826974. , a substantial increase, for example at 25. 〇 or 40. In the month, the ratio of the cups from H t I r 1 to the number of antibodies in the preparation was small. The lyophilized preparation of the present invention <w <RTIgt; is that the amount of the polymer formed by the antibody during storage does not increase. The content of the polymer and the content of the decomposition product can be determined by size exclusion chromatography, for example, by deionization, for example, by ion exchange chromatography; the content of the oxide can be determined, for example, by hydrophobic HPLC.

The "therapeutically effective amount" of the antibody of the present invention means an amount of the antibody which is effective for the prevention or treatment of a disease which can be effectively treated. The so-called "disease" is any symptom that is profitable by treatment with antibodies.纟 Includes: Chronic and acute diseases or diseases including the pathological state of V-feeding animals. The therapeutically effective amount of the antibody present in the formulation can be determined, for example, by considering the desired use 1 and the form of administration. Exemplary antibody concentrations in the formulation range from about 1 mg/mL to about 200 mg/mL, preferably from about 5 mg/mL to about 50 mg/mL, most preferably about 1 〇 (5) "(5) B and / Or about 2 〇mg/mL. The so-called "antibody" contained in this patent is used in the broadest sense, including, in particular, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, or as long as the desired biological activity is maintained. Antibody fragments can be included. Further, the antibody 'included in the present invention' is not particularly limited as long as it can bind to a desired antigen, and can be suitably used: a mouse antibody, a rat antibody, a rabbit antibody, a sheep antibody, a camel antibody, a chicken antibody, a chimeric antibody. , human antibodies, human antibodies, etc. The antibody may be a plurality of antibodies or a monoclonal antibody. The present invention relates to a freeze-dried pharmaceutical preparation which is stable with an antibody. The antibody in the preparation can be prepared by using a technique known in the art for producing antibodies and being used in the art as 124402.doc -18-200826974. Examples of the method for producing an exemplary monoclonal antibody include: (1) purification of a biological polymer used as an immunogen and/or production of cells overexpressing antigenic proteins on a cell surface; (2) The antigen is injected into the animal cell for immunization, and then the blood is collected and the antibody price is determined to determine the time at which the spleen is removed, and the process of preparing the antibody-producing cells; (3) preparation of myeloma cells; (4) The antibody-producing cells are fused with the cells of the myeloma cells; (5) for the production of the target (the selection of the fusion group of cardiac antibodies; (6) the isolation into a single cell strain (selection); (7) as appropriate for a large number of Production of fusion tumors of monoclonal antibodies or breeding of animals for transplantation of fusion tumors; (8) Selection of genes encoding human monoclonal antibodies from antibody-producing cells such as fusion tumors, and recombining them into appropriate vectors Then, it is introduced into a host (for example, a mammalian cell strain, Escherichia coli, yeast cells, insect cells, plant cells, etc.) to prepare a recombinant antibody produced by genetic recombination technology. (9) Purification of antibodies obtained in such a manner; (10) Studies on the G-activity and specificity of recognition of monoclonal antibodies produced in such a manner, or analysis of characteristics as a marker drug Further, a plurality of antibodies can be produced in the same manner as those known to the manufacturer. The antibodies contained in the patent also include: among the amino acid sequences of the heavy and/or light chains of the antibody, An antibody comprising one or more amino acids which produces a heavy chain and/or a light chain of a deletion, substitution or addition of an amino acid sequence. In the amino acid sequence of the antibody of the invention, such as the aforementioned amine group Partial changes (deletions, substitutions, insertions, additions) of the acid can be introduced by partially altering the base sequence encoding the amino acid sequence. The base sequence of the base sequence is changed to 124402.doc •19-200826974, The known site-specific mutagenesis can be used and introduced by a general method (Proc Natl Acad Sci USA., 1984, Vol. 181 · 5662). The antibodies contained in this patent are also included. Any of the immunoglobulins and antibodies having the same type. The antibody also includes a modified antibody that is recombined into the subtype, or a modified antibody that further changes the heavy chain immobilization region. Further, it also includes iodine, bismuth, indium, JC Goding, Monoclonal Antibodies: principles and practice, 1993 Academic Press, such as Pseudomonas aeruginosa toxin, diphteria toxin, ricin, bacterial toxins, and killing Chemotherapy agents such as cancer (methotrexate), mitomycin, and calicheamicin (DJKing, Applications and Engineering of Monoclonal Antibodies, 1998 TJIntenational Ltd.; MLGrossbard, Monoclonal Antibody-Base Therapy of Cancer · 1998 Marcel Dekker Inc), and further with prodrugs such as Maytansinoid (Chari et al, Cancer Res., 1992 Vol. 52: 127; Liu et al. 5 Proc. Natl. Acad. Sci. USA, 1996 Vol 93: 8681) and the like, thereby further enhancing the therapeutic effect on diseases such as cancer. Further, the antibody encompassed by this patent also includes a functional fragment of the antibody. The term "functional fragment" refers to a part (partial fragment) of an antibody, and means that one or more antibodies are allowed to act on an antigen, and specifically, F(abf)2, Fab', Fab, Fv, Disulfide-bonded Fv, single-chain Fv (scFv), bifunctional antibody, and the like (D. J. King, Applications and Engineering of Monoclonal Antibodies, 124402.doc -20- 200826974 1998 Τ· J. International Ltd) The antibody contained in this patent is a plurality of antibodies or/and monoclonal antibodies, preferably human antibodies, humanized antibodies or chimeric antibodies. The antibody is preferably IgG, and the subtype of IgG is preferably any of IgG1, IgG2, and IgG4. Further, the IgG may be an IgG 〇 which is genetically altered by a part of the amino acid sequence of the immobilization region, and which is deleted and/or substituted and/or inserted by the amino acid, and more preferably, the international publication The anti-HLA-DR human monoclonal antibodies described in WO2003/033538 are HD3, HD4, HD6, HD7, HD8, HD10, HD4G1, HD4G2Ser, HD4G4, HD8G1, HD8GlSer, HD8G2, HD8G2Ser, and HD8G4. Among them, the fusion tumors of HD4, HD6, HD8 and HD10 were produced, which were numbered by FERM BP-7771, FERM BP-7772, FERM BP-7773, and FERM BP-7774, respectively. On October 11, 2001, the international pinned on The patented bio-receiving center of the Industrial and Technological Research Institute of the National Institute of Industrial and Technological Affairs (the sixth in the center of the 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, In addition, the fusion tumors of HD3 and HD7 were produced under the numbers FERM BP-08534 and FERM BP-08536, respectively, and the international center was placed at the same center on October 31, 2003. Further, the present patent also includes a human monoclonal antibody against CD40, and preferably an anti-CD40 antagonist antibody described in International Publication No. WO2002088186, that is, KM281 small 10, KM281-2-10-1-2, KM283. -5, KM225-2-56, KM292 small 24, KM341-6-9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417, F4-465, and anti-CD40 agonistic antibodies ie KM302-1, KM341 small 19 , KM643-4-11, 2053, 2105, -21- 124402.doc 200826974

3821, 3822, 285, 110, 115, F1 - 102, F2-103, F5-77, F5-157. Among them, the fusion tumor strain 〖]^302-1, 1(:]^281-1_10, 尺]^281-2-10-1-2, was based on FERM BP-75 78, FERM on May 9, 2001 BP-7579, FERM BP-7580, KM341-1-19 and 4D11, on September 27, 2001, ij with FERM BP-7759, FERM BP-7758, strain 2105 in 2002 On April 17th, the number of the FERM BP-8024 is based on the Budapest Treaty and is internationally affixed to the Patent Bio-Receiving Center of the Institute of Industrial Technology, the Independent Administrative Corporation (Tsukichi City, Tsukuba, 1st, 1st, 1st, 1st, Central). The plastids of the variable regions of the heavy and light chains of F2-103, F5-77 and F5-157 were based on ATCC PTA-3302 (F2-103 heavy chain) and ATCC PTA- on April 19, 2001. 3303 (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) No., fusion tumor strains F1-102 and F4-465, on April 24, 2001, under the number of ATCC PTA-3337 and ATCC PTA-3338, based on the Budapest Treaty and internationally (American Type Culuture Collection, 10801 University Blvd., Manassas, Virginia, US A) (National Bacterial Culture Collection, Manilas 10801 University Boulevard, Virginia 20110-2209). The lyophilized formulation of the present invention, using the above Any of the antibodies is stable. The lyophilized preparation of the present invention can be prepared by preparing a solution preparation containing the above antibody and various additives as an active ingredient, and then lyophilizing the solution, according to a preferred embodiment of the present invention. Morphological, lyophilized preparation, except for antibody 124402.doc -22- 200826974 'also contains polyol, surname θ active agent. ~ 曰 物质 substance' and then contains buffer and interface 7; bundled dry lyophilized "" and "cooling; Dongganyu (four) called Na" means that the substance to be dried is firstly cooled, followed by ice or cold; the solvent of East is sublimated in a vacuum environment and is removed. The stability of cold-dried products, dried in cold

Then, "I may also contain an excipient. Cold; the east drying step can be carried out by a known method. The present invention includes the "Jiankouyue Additive", which includes the freeze-dried preparation other than the active ingredient. All of the components include, for example, a buffer, a pH adjuster, an isotonic agent, an excipient, a stabilizer, a surfactant, a preservative, and the like. Further, a combination of 4 k 仏 and one additive component exhibits two or more effects. "Buffer" means an additive which stabilizes the pH change by the action of the acid-base component. The buffer concentration, depending on its buffering capacity and/or the desired osmotic pressure, is about ( 杂 to (10) 匪 ^, preferably from j mmol / L to 20 mm 〇 i / L, and most preferably about 1 〇_〇1/L. A preferred buffer, & a faceted amine salt (such as sodium glutamate), or #acid and other organic acid buffers. The best is the face amine salt. "Inhibitor" is a substance having a plurality of hydroxyl groups and is also used as an excipient in a lyophilized preparation. "Polyol" includes: sugar (reduced and non-reducing sugar) sugar alcohols, and sugar acids. Here, preferred polyols have a molecular weight of less than about 600 KD (e.g., in the range of from about 12 KD to about 4 KD). "Reducing sugar" is a semi-acetal group which can reduce metal ions or have a co-reaction with the amine 124402.doc -23-200826974 acid and other amine groups in the protein; "non-reducing sugar" Those who do not have the properties of reducing sugar. As examples of reducing sugars, there are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose, and glucose. As examples of non-reducing sugars, there are strict sugar, trehalose, sorbose, raffinose, and (iv) sugar. Examples of the sugar alcohols are mannitol, xylitol, erythritol, threitol, sorbitol, and glycerin. Sugar acids include L-gluconic acid and its metal salts. The reducing sugar is used in cold; the east dry preparation is not suitable for promoting the formation of oxidants. Further, in the case of using a polyol for protecting the protein against the pressure accompanying the cold and drying, it is preferable to form an amorphous structure when performing cold bridging and drying. As for the non-opening of the amorphous structure + W U7, the case of the sputum is sucrose, sucrose and so on. The present invention - a non-reducing sugar which is non-reducing sugar and which is amorphous or non-reducing sugar and which is an amorphous sugar or a sugar alcohol. More 妒θ— , w 4 糖鲜寺 Better 疋庶 sugar, trehalose sorbose

L, such as raffinose, is a non-reducing sugar and is amorphous ^-sugar, and sugar and trehalose. Also, ^ is the best one. The cold-dried preparation of the invention may also contain a plurality of so-called "crystallized kelp repellers, spoons and scallops", and if it is frozen, it becomes a crystallized structure: crystallinity such as amino acid, glycine acid, etc. Amino acid and the like. = 疋 alanine, 1 search especially good mg / mL, better ^ 5 40 mg / mL, preferably 10 ~ 30 polyol and crystal Γ μ ~ Zeng #, especially good 20_祉. use. In the present invention, 1' can be used as an excipient or an isotonic agent as a "so-called "excipient"" and has the function of maintaining the shape of a cake of a dry preparation of 124402.doc -24·200826974 and ή ua It is a general-purpose, is ^ y-pd, ritual sugar used as an excipient, in addition to polyterpene alcohol and a crystalline substance, for the function of the protein to be used for freezing and drying. Excipients provide enhanced properties when enhanced protein stability during long-term preservation. In addition, the four substances can also function as isotonic agents Γ, 敫, and r. "Isotonic agent" refers to a method that modulates the osmotic pressure so that the subject preparation has soil, eight blood, and the same osmotic pressure in the night. The special osmotic preparation has about 25 〇 to

The osmotic pressure of m 〇 sm / kg is preferably an osmotic pressure ratio of osmotic pressure of physiological saline to 1. In the present invention, the alanine used as an excipient functions to maintain a cake shape, and the polyol acts to protect the protein against the force accompanying cold rolling and drying. In the invention, the silk polyol is also used as an anti-agglomerating agent, and it plays an important role in shortening the recovery time of the cold-drying preparation in the solution of the non-granular substance. The polyol ' is added in an amount that varies depending on the osmotic pressure desired for the formulation. Preferably, it is 'cooled after reconstitution; the east dry preparation is isotonic, but it may also be south or low permeability. Surfactants include nonionic surfactants such as polysorbates (e.g., polysorbate 2, 80, etc.) or P〇L XAMER (e.g., p〇L XAMER (8)). The amount of surfactant added is such that the agglutination of the formulated antibody and/or the granules in the formulation and/or the reduction of protein adsorption on the container are reduced. Preferably, it comprises a polysorbate, and most preferably a polysorbate 8 is used as a surfactant. The surfactant is preferably present in the formulation in an amount of from 0.02 11 / 1111 ^ to 〇 j mg / mL, preferably in an amount of about 5 mg / mL. 124402.doc -25- 200826974 As an example of a composition prior to lyophilization, the alpha butyl system comprises from about 1 mg/mL to

6〇mg/mL IgG antibody, about 1〇mm〇1/L main 20mmol/L sodium glutamate (pH 5.5), about 〇.〇5 mg/mL of poly mountain plow stuffing And a preparation of 20 mg/mL alanine and 5 mg/mL to 5 〇 (4) 斧 axe as an excipient. The above-mentioned cold; the dried sorghum is made into a dry powder by cold slag drying, which can be easily restored into a solution containing no granules suitable for administration to humans.

In the present invention, a crystalline substance f (preferably alanine) is added to the antibody, and the non-reducing sugar and the amorphous polyol (preferably heart and trehalose) are used as excipients for freezing. Drying, thereby providing a stable frozen soft dry preparation. "Upper" can be obtained by including a crystalline phase of a crystalline material: a structure: and comprising an antibody and a non-reducing sugar and/or an amorphous polyol (preferably a sugar and trehalose) The amorphous phase protects the protein from the pressure of cooling and drying, thereby obtaining a stable freeze-dried preparation. The ratio of the alanine to the silk and the blending amount of the excipient used in the present invention can be based on the activity The concentration of the antibody of the component is appropriately adjusted. The ratio of the ratio of the sugar of the vegetable sugar to the alanine (the mass of the sucrose/the mass of the alanine) or the concentration (the sucrose concentration/alanine concentration) is set in the cold-dried formulation. When the raspberry sex is good, R is 0.25 or more, preferably 〇5 to 25, more preferably 1 to 2', and the best is 1.5. 'And' is to set Q as a cold-drying preparation. When the mass ratio of the antibody to the alanine (antibody mass/alanine mass) or the concentration ratio (antibody concentration/alanine concentration) is present, the cold sugar/alanine cold is used to dry the preparation in a ratio of R=l.5. The Q with good stability is 〇·05 to 3, preferably 〇〇5 to 15. 124402.doc -26- 200826974, a stable cold 4 dry asexual agent containing antibody, alanine, and sucrose in the combination of R and Q described above, the antibody mass and the quality of alanine and the quality of sucrose The ratio of the antibody mass to the total of prisin and sucrose is ~. The freeze-dried preparation of the present invention is used after being restored. The term "recovery" means that the lyophilized preparation is rehydrated in a solution to form a clarified solution free of particles; the "recovery time" is the time required for C]. An important feature of the cold-to-east dry preparation is that the time required for the recovery time s of the product and the re-X σ is required to be short. In order to be rehydrated very quickly and completely, it is important to have a cake having a highly porous structure. The cake structure is a function of the parameters of the protein concentration, the type and concentration of the excipients, and the process parameters of the cold j drying cycle in (iv). In general, if the protein concentration increases, the recovery time increases. Therefore, when the recovery time is long, the protein is deteriorated due to the protein being exposed to the further concentrated solution for a longer period of time. Furthermore, from the point of use of hydrazine, until the product is completely rehydrated, it can be administered by ensuring that the product is completely rehydrated, thereby ensuring that the product does not contain granules, is administered in the correct amount, and is not sterilized. Affected. That is, rapid rehydration is beneficial to both the patient and the doctor. The solution formulation ' can be cold-dried using appropriate drying parameters. Cold to dry the sword, can maintain the stability of the biological activity of the antibody, and protect the antibody for the purpose of the human body to be tested, so that it will not be biologically and chemically decomposed in the final product. The cold-dried preparation is rehydrated with a diluent (for example, water for injection, 124402.doc -27-200826974, to provide a solution free of granules. The reconstituted antibody solution, even at ambient temperature) After the long-term storage of the freeze-dried cake, there will be no granules. The reconstituted solution can be administered parenterally, preferably intramuscularly or intramuscularly or subcutaneously, to the test subject. The composition ensures that the formation of protein agglomerates and granules in the immunoglobulin-containing reagent is minimized, and the antibody in the solution maintains its immunological activity even over time. The composition includes: neutral or acidic PH value buffer contains antibody, alanine, poly(iv) and surfactant, which is water-cooled; aseptic medicinal preparation prepared by (4) before drying in East; The substance may further contain excipients and/or isotonic agents other than alanine and a polyhydric alcohol.

The preparation may be administered to a mammal, preferably a human, in a known manner, for example, as a globule, or intravenously administered intramuscularly, intraperitoneally, intracerebally, within a certain period of time. , subcutaneous, intracardiac, intratracheal, intraarachnoid, oral, topical, or via: infusion. In a preferred embodiment, the formulation can be administered to a mammal by intravenous administration. To achieve this (4), the formulation is injected, for example, using a syringe or via a drip tube.实施 Embodiments The present invention will be described in more detail with reference to the following embodiments without restricting the invention. For example, the invention can be fully understood. However, the scope of such inventions. Based on the description of the present invention, the modifications and modifications are also included in the present invention. The combination of alanine and various excipients of Example 1 is used in the case of the above-mentioned invention. 124402.doc -28 - 200826974, in the case of International Publication No. WO2003/033538 The antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described above was used as an antibody to prepare a freeze-dried preparation. In this example, the formulations shown in Table 1 were prepared and a detailed study was conducted with suitable excipients in combination with alanine. [Table 1] List of preparations for use in this example Antibody concentration buffer Excipient surfactant pH 1 10 mg/mL Anti-HLA-DR antibody 10 mmol/L Glutamate 20 mg/mL Alanine ^^ 0.05 Mg/mL Polysorbate 80 5.5 2 30 mg/mL Trehalose 3 Sucrose 4 Preparation of sodium chloride (1) preparation sample The test used in this study was an anti-HLA-DR antibody (about 20 mg/mL). According to the method described in International Publication No. WO2003/0033538, at the Institute of Pharmaceutical Production Technology of Kirin Brewery Co., Ltd. (after July 1, 2007, at the Institute of Production Technology of Kirin Pharmaceutical Co., Ltd., Japan) I) L-glutamate monohydrate (Japanese Pharmacopoeia prescription), alanine (Ala, Japanese Pharmacopoeia), trehalose (Tor, Japanese Pharmacopoeia), sucrose (Sue, Pharmacy) Fang), sodium chloride (NaC 曰 曰 曰 曰 )), hydrochloric acid (Japanese Pharmacopoeia), polysorbate 80 (Japanese Pharmacopoeia), water for injection (曰 药 局). Each of the preparation samples was prepared by previously preparing a pseudo drug solution containing no original drug, and replacing the anti-HLA-DR antibody solution with the preparation pseudo drug solution using a NAP column (Pharmacia Biotech). Further, the protein concentration was adjusted by using the OD280 nm absorbance coefficient ε=1.4 for 124402.doc -29-200826974, and the concentration was adjusted. Each of the preparation samples was sterile-filtered using a 0.22 μηχ filter (manufactured by MHhpore) in a Clean Bench, and each 1 mL of the filtrate was filled in a 5 mL glass vial (suitable for the Japanese Pharmacopoeia). After filling, it was semi-capped with a rubber stopper, and cold-cooled using a cold beam dryer manufactured by Sakamoto Vacuum Technology Co., Ltd.; In addition, in a sterile dust-free workbench (4) 0.22 μηι bottle cap filter (Nalgene), the anti-HLA_DR antibody-containing solution (pseudo-pharmaceutical solution) used to dilute the analysis sample and the analysis blank is used for sterile filtration. (2) The test conditions were carried out in the present embodiment, and pressure was applied to each of the preparation samples under the following conditions for the stability evaluation. / Thermal stability test: an incubator with a temperature control of 40 < t or 25 < t (TABAI ESPEC) In the middle, save for months and 3 months. In addition, from the heat stress load to the start of the analysis, 'store each sample in a low temperature library controlled at 4 °C. (3) Analytical method Visual appearance ··· Under the light, the shape of the cake was observed with the naked eye, and the appearance was described. The following analysis was performed after visual observation, and then re-dissolved by adding j mL of water for injection, and then analyzed. Insoluble foreign matter and turbidity: white light source Below, at a position of about 5 〇〇〇 - to visually check for the presence or absence of insoluble foreign matter and turbidity. Size exclusion HPLC identification (SEC): polymer content and decomposition content, 12 4402.doc -30 - 200826974 is calculated by size exclusion high-speed liquid chromatography. The sample is diluted to 1 mg/mL as needed, and 20 μιη at ambient temperature. The separation system uses TSK gel G3000 SWXL 30 Cmx 7.8 mm. (TOSOH company) column, using 20 mmol / L sodium phosphate, 500 mmol / L sodium chloride pH 7.0 as the mobile phase, at a flow rate of 0.5 mL / min, analysis time of 30 minutes, at 215 nm The test will be carried out as a polymer before the main peak, and will be defined as a decomposition product after the main peak. Cation exchange HPLC identification (IEC): Deamination content using cation exchange high-speed liquid chromatography Calculated by injecting 60 pL into a sample diluted to 5 mg/mL. TSK gel BIO Assist S (manufactured by TOSOH) was used as a separation column and was detected at 280 nm. As a mobile phase, 20 mM tartaric acid was used for phase A. pH 4.5, for phase B using 20 mM tartaric acid, 1 Μ sodium chloride pH 4.5, using the most suitable gradient conditions for analysis. Furthermore, the degraded material that will elute in front of the main peak is defined as deammonium. (HIC) ·· The content of the oxidant was calculated by hydrophobic chromatography. The sample treatment solution (2 〇mm〇i/L sodium phosphate, 0.984 mol/L ammonium sulfate, 7.5 mmol/L methionine) was appropriately diluted into 20 pL. A sample of 1 mg/mL was used for analysis and detection at 215 nm using TSK gel Butyl-NPR (manufactured by TOSOH) as a separation column. The mobile phase system uses 2〇mmol/L sodium acetate and ρΗ 6·5 as phase A; using 20 mm〇i/L sodium citrate, 1.2 mol/L sulfuric acid, and ρΗ 6·5 as phase B, the most Analyze the gradient conditions as appropriate. Further, the deteriorated substance which is eluted in front of the main peak is defined as an oxide. Reduction SDS-PAGE Identification · The sample solution was diluted to 400 124402.doc -31 - 200826974 pg/mL as needed. SDS Sample Buffer (manufactured by Invitrogen), Sample Reducing Agent (manufactured by Invitrogen), and H2 oxime were placed in a sample solution, and heated at 95 ° C for 5 minutes to prepare a reduced sample solution. In the swimming turmoil, Tris-Glycine SDS Running buffer (Invitrogen) was used for electrophoresis. 5 μL of the sample solution was added to 4 to 20% Tris_Glycine Gel (manufactured by Invitrogen), and electrophoresis was carried out at a fixed voltage of about 125 V until the blue color of bromophenol blue contained in the sample solution was moved to the vicinity of the lower end of the gel. The gel after the completion of the electrophoresis was subjected to silver staining and detected. Further, in order to determine the approximate molecular weight of the sample and the deteriorated body, molecular weight markers (including proteins of 97 KDa, 66 KDa, 45 KDa, 30 KDa, 20 KDa, and 14 KDa) were simultaneously subjected to electrophoresis. Non-reducing SDS-PAGE identification: Dilute the sample solution to 400 pg/mL as needed. LInto the sample solution, insert LDS Sample Buffer (Invitrogen) and H20 to prepare a non-reducing sample solution. The electrophoresis was filled with Tris-Acetate SDS Running buffer (manufactured by Invitrogen). 5 pL of the sample solution was supplied to a 3 to 8% Tris-Acetate Gel (Invitrogen), and electrophoresis was carried out at a fixed voltage of about 125 V until the blue color of the bromophenol blue contained in the sample solution was moved to the lower end of the gel. Pay close. The gel after the completion of the electrophoresis was subjected to silver staining and detected. Further, in order to determine the approximate molecular weight of the sample and the inferior body, the molecular weight markers (including 220 KDa, 116 KDa, 97.4 KDa, 63.3 KDa, 54.4 KDa, 36.5 KDa protein) were simultaneously subjected to electrophoresis. Osmotic pressure ratio: The osmotic pressure measurement was carried out using an automatic osmotic pressure measuring device (OSMO STATION OM-6050, manufactured by Arkray Co., Ltd.), and the ratio of the water osmotic pressure of 124402.doc - 32 - 200826974 to the measured physiological salt was calculated. ρ Η value: The pH value was measured using an automatic enthalpy measuring device (METTLER T〇LEDc^v system Μρ·23〇, etc.). At the beginning of the measurement, the standard solution having a positive value of 4, a positive value of 7, and a value of 9 was used for the calibration, and then the measurement was carried out. The water-jet test was carried out, and the moisture content was measured using a trace moisture measuring device (manufactured by Hiranuma Sangyo Co., Ltd., LE-20SA) connected to an automatic moisture gasification device (manufactured by Hiranuma Sangyo Co., Ltd.) to calculate the moisture content. ο υ (4) Results and investigation Fig. 1 shows changes in the polymer when each lyophilized preparation was stored at 25 t and 4 Torr. The amount of polymer was determined by size exclusion chromatography. In the preparation containing only alanine as an excipient, it was confirmed to be stored at 4 Gt for 2 months, and it was confirmed that the polymer was increased by about 5%, and after 3 months of storage, it was confirmed that the polymer increased by about 9%. 1 Alanine and sodium chloride. In the preparation of the excipient, it was visually inspected by the naked eye after re-dissolution, and the increase in the foreign matter, turbidity, and daily polymer was also "unstable." In the preparations combined with trehalose and Wei, the increase in the polymer was not confirmed even after storage for 3 months at 40t, which was very high. It is clear that by incorporating the above-mentioned polyol (trehalose, ginseng) into alanine, the polymer is dramatically suppressed, indicating that the stability is high. In addition, in other analytical items (cakes, foreign bodies, turbidity, decomposition products, insoluble fine particles, deaminating substances, oxidants), a combination of alanine and a polyol was hardly found before and after applying a heat and pressure load. There are changes, for stability. According to the present embodiment, it is considered that the excipient which is a cold-dried preparation for tying the antibody is preferably a combination of alanine and a polyhydric alcohol. Preferably, 124402.doc -33- 200826974 is 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 is clear from Example 1. As the polyol in combination with alanine, trehalose or sucrose is preferred. In the present example, sucrose was used as a polyol, the concentration of alanine was fixed, and the sucrose concentration mixed with alanine was changed, and the optimum concentration ratio R of the inhibiting polymer was examined. Here, R represents the sucrose mass/alanine mass present in the freeze-dried product. A freeze-dried preparation was prepared by using an antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described in International Publication No. WO2003/033538 as an antibody. In the present example, the preparations shown in Table 2 were prepared, and a better concentration of sucrose combined with alanine was studied in detail. [Table 2] List of preparations for this example Antibody concentration buffer Excipient surfactant pH value R 1 10 mg/mL Anti-HLA-DR antibody 10 mmol/L Glutamate 20 mg/mL Alanine 50 mg /mL Sucrose 0.05 mg/mL Polysorbate 80 5.5 2.5 2 40 mg/mL 2 3 35 mg/ml 1.75 4 30 mg/mL 1.5 5 25 mg/mL 1.25 6 20 mg/mL 1 7 15 mg/mL 0.75 8 10 mg/mL 0.5 9 5 mg/mL 0.25 10 0 (1) Materials and Methods The materials and analytical methods used in this example were the same as those shown in Example 1 of 124402.doc-34-200826974. (2) Test conditions and examples! In the same manner as the method indicated, the thermal stability test was carried out by holding the temperature control at 25 C or 4 (TC incubator (TABAI _c) for ^month and 3 months. Again, after applying the heat and pressure load At the beginning of the analysis, each sample was stored in a temperature controlled sub-zero reservoir. (3) Results and investigation

Figure 2 shows the change in polymer when each lyophilized formulation was stored under 25 ounces of it. The amount of polymer was determined by size exclusion chromatography. It is clear that the polymer formation is inhibited as the amount of f added increases, and it is shown to be in the range of 25 to R = 2.5. Further, in the gamma analysis, a swimming image in which the same tendency as that obtained by size exclusion chromatography was observed was also obtained. In the present embodiment, the cake was formed at any concentration, especially in the form of a whole cake having R = 1.5 or less, which exhibited a shape which can withstand long-term storage. In addition, in other analysis items (foreign matter, turbidity, insoluble fine particles, de-alcohol, oxidized body), the change before and after the heat stress load was hardly confirmed, and it was stable. In the present implementation, it is clear that the optimum sucrose mass ratio in combination with alanine as the excipient of the cold-stable dry preparation for stabilizing the antibody is sorbose mass/alanine mass). The measure of good stability is 〇.25 or more, preferably = 〇.5 to 2.5, more preferably 丨 to 2, more preferably 〇.75 to 2. The best effect of the antibody concentration in Example 3 is 124402.doc-35-200826974. The antibody against HLA-DR (anti-HLA-DR antibody, IgG1) described in International Publication No. WO2003/033538 is used as an antibody. A lyophilized preparation was prepared. In the present example, the preparations shown in Table 3 were prepared, and the effects of the preparation of the sucrose/alanine prescription (R = 1.5) which stabilized the antibody obtained in Example 2 on the preparations having different antibody concentrations were carried out. Evaluation. Here, q is taken as the ratio of the mass of the antibody to the mass of the alanine. Namely, Q represents the mass of the antibody/alanine in the freeze-dried preparation. [Table 3] List of preparations for this example Antibody concentration Antibody type Excipient surfactant pH 〇1 60 mg/mL Anti-HLA-DR antibody 20 mg/mL Alanine 30 mg/mL Sucrose 0.05 mg/mL Polysorbate 80 5.5 V 3 1.5 0.5 0.05 2 30 mg/mL 3 10 mg/mL 4 1 mg/mL 5 10 mg/mL 0.5 -36 - 200826974 = 2: The amount of polymer was determined by size exclusion chromatography. The compound (4) which was resistant to two degrees (the most antibody amount) was kept for 3 months and the polymer was about 2%. In general, when the concentration of an antibody becomes high, the amount of polymer produced increases, and it becomes unstable. The antibody concentration of the right example i is 1 、, and the apricotic acid is separately prescribed: L can be clearly I. The amount of polymer after 3 months of storage: :, ".贞 can sufficiently inhibit polymer formation. Furthermore, the formulation in which the antibody concentration is the lowest concentration (the minimum amount of antibody) iQ = 〇. 〇 5 is almost undefined: the change before and after the application of the pressure load is stability. Further, in the other analysis items (foreign matter, turbidity, decomposition product, deamamine, oxidized body), almost no change was observed before and after the application of the pressure load, and it was stable. In the present embodiment, it is understood that a stable freeze-dried preparation is obtained by containing alanine/sucrose in a ratio of R = 1.5 with respect to Q = 〇〇 5 to 3. That is, it is disclosed that the stable freeze-dried preparation is freeze-dried in a mass ratio of the mass of the antibody to the three components of the alanine and the sucrose mass of 1:1·3~2〇:〇·5~3〇. Obtained in the form of a preparation. In the case where the ratio of the antibody mass to the total of the mass of the alanine and the mass of the sucrose is expressed (the mass of the antibody is set to 1) '1:5/6 to 50, preferably 1:1 to 2 Å, Better is 1:1~10, especially good 1:5. Example 4 Study using other antibodies In this study, the best alanine/sucrose prescription (RU) for the stabilization of the antibody obtained in Example 2 was used for the cd4 disclosed in International Publication No. WO05563981. The recombinant human IgG4 antibody 4D11 (anti-CD40 antibody) was recombined to study the stability of the preparation. 124402.doc 37 200826974 Formulations shown, evaluation of antibody species In this example, the effects of the formulations shown in Table 4 on the stability of the formulation were determined. [Table 4] For the purpose of this embodiment

(1) Materials and methods

The material used in this embodiment is the same as the one shown in the example. R = 1.5, q = 0.5, and the ratio of alanine and Yan sugar mass to antibody mass is 1:5. (2) The test conditions were stored in the same manner as in the method disclosed in Example 1 at a temperature controlled at 25 C or 40 ° C in a culture of WTABAI ESpEC) for one month and three months, and only the heat stability test was carried out. . Further, from the application of the heat and pressure load to the start of the knife-cutting, each sample was stored in a temperature-controlled low temperature reservoir. (3) Results and investigations Figure 4 shows the culture of the polymer when each lyophilized preparation was stored at 40 ° C (initial ratio was obtained by dividing the amount of polymer before lyophilization, before lyophilization) The value is set to 1). The amount of polymer was determined by size exclusion HPLC. Among the anti-CD4 〇 antibodies, in the preparation using alanine and sucrose as excipients, it was confirmed that the inhibition of the formation of the polymer during storage was independent of the degree and time, and the antibody in the preparation was stably maintained. Further, in other analysis items (foreign matter, turbidity, decomposition product, deaminating amine, oxidized body), it was hardly confirmed that the change of 124402.doc -38-200826974 before and after the application of the heat stress load was stability. In the present example, it was found that a stable freeze-dried preparation using alanine and a recommendation sugar as an excipient is stable regardless of the type of the antibody. All publications, patents and patent applications cited in this specification are hereby incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is shown at 25. (: or 40. (;; the amount of change in the amount of polymerization enthalpy when each lyophilized preparation was stored. The amount of polymer was determined by size exclusion HPLC. When alanine was used alone, the polymer formation was marked, but with alanine The preparation with the combination of trehalose and/or sucrose is diazepam. Fig. 2 is a diagram showing the wheatification of the polymer oxime when each lyophilized preparation is stored at 25t: or 4 (rc). The amount of the polymer is determined by size exclusion HpLc. The formulation having a ratio of alanine to the combined sucrose of 〇25 to 2.5 showed stability. Figure 3 shows the amount of polymeric chelating agent when each lyophilized preparation was stored at 25 ° C or 40 ° C. The graph of the change. The amount of polymer was measured by size exclusion HPLC. The formulation of Q (the ratio of antibody mass to alanine mass) of 0 05 to 3 showed stability. - Figure 4 shows the preservation at 251 or 4 (rc) The graph of the polymerization and the amount of change in each freeze-dried preparation. The amount of the polymer was measured by size exclusion hplc. In the anti-HLA-DR antibody and anti-CD40 antibody, the freeze-dried preparation with alanine and sucrose as excipients showed For stability. 124402.do c -39-

Claims (1)

200826974 X. Patent application scope: 1. A lyophilized preparation containing an antibody, which further contains alanine and a polyhydrazine f* as an excipient. Wherein the polyol is a non-reducing sugar, wherein the polyol is a non-reducing sugar. 2. The lyophilized preparation of claim 1 and which is an amorphous polyol. 3. The lyophilized preparation of claim 1 which is an amorphous sugar. 4. The lyophilized preparation of claim 3, wherein the polyol is sucrose or trehalose. The cold lyophilized preparation according to any one of items 1 to 4, wherein the alanine of the antibody Berry 1 is The quality of the polyol is . 6. The freeze-dried preparation according to claim 5, wherein the ratio of the mass of the polyol to the mass of the alanine is from 0.5 to 2.5. 7. The cold-dried preparation according to claim 5 or 6, wherein the ratio of the mass of the antibody to the mass of the alanine is 0.05 to 3. 8. According to the requirements of items 1 to 7 - 冷冻10, human + t, the lyophilized preparation, wherein the mass ratio of the antibody mass to the mass of the alanine and the 3 components of the axe #田庶檐 quality is 1··· 3~20: 0.5~30. 9. The freeze-dried preparation of human soil _ ^ according to any one of claims 1 to 8, which further comprises a buffer. 10. The freeze-drying method of claim 9, wherein the buffer is a face acid buffer 0 11_freely dried i 〇 mM as claimed in claim 9 or 10. , a sputum preparation, wherein the concentration of the buffer is 124402.doc 200826974 12 · as claimed in claim 1 ... the cold-dried preparation, which further contains a surfactant 〇 13 · The lyophilized preparation of claim 12 The purpose of the alcohol is g. "The medium surfactant is polysorbate. 14. The lyophilized preparation according to any one of claims 丨 to 13, which is a pharmaceutical preparation containing an antibody as an active ingredient. The method for drying φ comprises: freeze-drying in an antibody/4, adding alanine and a polyhydric alcohol, wherein the polyol is 16. The method for producing a freeze-dried preparation according to claim 15 is a non-reducing sugar and is amorphous. The polyol is 17. The polyol is 17·cool as claimed in claim 15; the method for producing the eastern dry preparation is non-reducing sugar and is amorphous sugar. The polyol is 18. The lyophilized preparation according to claim 17 The method of sucrose or trehalose. / 19. The preparation method of the lyophilized preparation according to any one of claims 15 to 18, the propylene 5/6~5Q relative to the antibody quality. And the mass of the lyophilized alcohol is 20. The ratio of the amount of the freeze-dried preparation / the mass of the alanine of claim 19 is .5 to 25. "Method, wherein the polyol is 21. The cold as in claim 19 or 20; The ratio of the quality of the East Dry Preparation to the mass of alanine is 0·0. 5 to 3. U. The method of producing a cold-dried preparation of mussels according to any one of claims 15 to 21, wherein the mass ratio of the antibody to the alanine is 1: 〇·3~5·30. , 庶 ^ set the quality of the 3 components 124402.doc