WO2008029908A1

WO2008029908A1 - Stable lyophilized pharmaceutical preparation comprising antibody

Info

Publication number: WO2008029908A1
Application number: PCT/JP2007/067481
Authority: WO
Inventors: Takahiko Ito; Tomoyoshi Ishikawa; Chigusa Suzuki
Original assignee: Kyowa Hakko Kirin Co., Ltd.
Priority date: 2006-09-07
Filing date: 2007-09-07
Publication date: 2008-03-13
Also published as: JPWO2008029908A1; TW200826974A

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

Specification

Stable lyophilized pharmaceutical formulation containing antibody

Technical field

[0001] The present invention relates to the field of antibody pharmaceutical formulations. Specifically, the present invention relates to a stable lyophilized pharmaceutical preparation containing an antibody.

Background art

[0002] With recent progress in biotechnology, 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 complex three-dimensional structures. Therefore, in order to preserve such proteins stably, the physical properties must be considered. Special technology is required. Formulations that keep proteins stable need to protect many different functional groups in proteins and maintain higher-order structures that are involved in activity. In the field of protein pharmaceuticals, The development of stable formulations that maintain both stability and activity is a major challenge.

[0003] Antibodies are one of the proteins useful for medical use. In recent years, monoclonal antibodies and polyclonal antibodies have been used for human clinical trials, aiming at treating various diseases such as antitumor, autoimmune diseases and infectious diseases. It is attracting attention in the pharmaceutical field! However, purified monoclonal antibodies and polyclonal antibodies are recognized as having the property of easily forming aggregates and granular insoluble particles in solution. It has become a problem. Stabilized antibody formulations that do not have such undesirable properties are desired.

[0004] As one of the methods for stabilizing an antibody, for example, as disclosed in WO098 / 56418 pamphlet, a therapeutically effective antibody that has not been previously lyophilized and an acetate buffer maintained at PH 5.0. It describes an aqueous pharmaceutical formulation containing an agent, a surfactant and a polyol. Solution formulations are easy and most economical to handle for manufacturers, and are the easiest to administer to patients. In general, however, the effects of chemical degradation (deamidation or oxidation) and physical degradation (aggregation and precipitation) in solution formulations Therefore, precise control of storage conditions is necessary to maintain stability, and low stability! Can be a drawback for antibodies for commercial applications.

[0005] In contrast, lyophilization of a product that is relatively unstable in solution may result in a stabilized product and thus has a longer shelf life and stability than a solution formulation. Is known in the art. (See Non-Patent Document 1). In the dried solid, the decomposition reaction is a force that can be 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 process requires Freeze-drying, which causes the ice to sublime from the frozen solution, leaving a solid, dried component of the original liquid. 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. It has a number of advantages. In addition, the lyophilized product can usually be rapidly dissolved and easily reconstituted prior to administration to a patient.

[0006] However, in order to achieve a lyophilized product with the desired characteristics, appropriate care must be taken in choosing the 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, excipients should be selected that ensure long-term stability with the dried solid.

[0007] The following documents are related to lyophilized preparations containing antibodies.

[0008] Patent Document 1 discloses a freeze-dried preparation containing an antibody, a sugar or amino sugar, an amino acid, and a surfactant. However, it is suggested that a freeze-dried preparation consisting of a combination of alanine and polyol is suitable.

[0009] Patent Document 2 discloses a freeze-dried preparation consisting only of protein, buffer, alanine and mannitol! However, it is suggested that a freeze-dried preparation consisting of a combination of alanine and amorphous polyol is suitable.

[0010] Patent Document 3 discloses a stable lyophilized preparation using 1 to 20 mg of D-mannitol per 1 mg of human monoclonal antibody. It is also preferable to use a lyophilized preparation composed of a combination of alanine and amorphous polyol. Also present a suitable mixing ratio.

[0011] There are also many scientific literatures on the effects 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 is not disclosed according to a set of generalized properties, and conflicting results will be seen depending on the protein studied and the amount of excipient used. It was.

[0012] Furthermore, the freeze-dried preparation disclosed in the existing patents causes generation of foreign substances, an increase in the polymer, an increase in the oxidant, and the like, and a stable preparation could not be found. For example, as disclosed in Patent Documents 4 and 5, freeze-dried 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.

Patent Document 1: International Publication No. W098 / 22136A2 Pamphlet

Patent Document 2: Pamphlet of International Publication No. W097 / 17064

Patent Document 3: Japanese Patent Laid-Open No. 5-25058

Patent Document 4: Japanese Translation of Japanese Patent Publication 3-504605

Patent Document 5: International Publication W089 / 11297 Pamphlet

Patent Document 1: “Remington's Pnarmaceutical Sciences” 15 / ¾¾, McKnouno, Mack Publishing Co., Easton, Philadelphia, pp 1483-1485 Disclosure of Invention

Problems to be solved by the invention

[0013] An object of the present invention is to provide a stable lyophilized pharmaceutical preparation containing an antibody. Specifically, an object of the present invention 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 an amorphous polyol. Means for solving the problem

[0014] The present inventors have conducted extensive research to obtain a stable lyophilized preparation containing a therapeutically effective amount of antibody. The result is a therapeutically effective amount of antibody plus non-reduced crystalline material. It has been found that a lyophilized preparation can be obtained by containing a polyol that is a sugar and an amorphous substance, and has an effect of suppressing the formation of a polymer generated during a storage period that is not found in a conventionally known lyophilized preparation. The present invention has been completed.

That is, the present invention is as follows.

[0016] [1] A freeze-dried preparation containing an antibody and further containing alanine and polyol as excipients.

[0017] [2] The freeze-dried preparation according to [1], wherein the polyol is a non-reducing sugar and is an amorphous polyol.

[0018] [3] The lyophilized preparation according to [1], wherein the polyol is a non-reducing sugar and is an amorphous sugar.

[4] The freeze-dried 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.

[0021] [6] The freeze-dried preparation according to [5], wherein the polyol mass / alanine mass ratio is 0.5 to 2.5.

[0022] [7] The freeze-dried preparation according to [5] or [6], wherein the antibody mass / alanine mass ratio is 0.05 to 3.

[0023] [8] The ratio of the mass of the three components of antibody mass, alanine mass, and sucrose mass is 1: 0.3 to 20: 0.5

The freeze-dried preparation according to any one of [1] to [7].

[0024] [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.

[0026] [11] The freeze-dried preparation according to [9] or [10], wherein the buffer solution has a concentration of 10 mM.

[0027] [12] The lyophilized 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 freeze-dried preparation according to any one of [1] to [; 13], which is a pharmaceutical preparation containing an antibody as an active ingredient.

[0030] [15] A method for producing a stable freeze-dried preparation comprising adding alanine and polyol to an antibody and freeze-drying.

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

[0032] [17] The method for producing a freeze-dried preparation according to [15], wherein the polyol is a non-reducing sugar and is an amorphous sugar. [0033] [18] The method for producing a freeze-dried preparation according to [16], wherein the polyol is sucrose or trehalose.

[0034] [19] Mass force S of alanine and polyol for antibody mass 1 is 5 / 6-50 [15]-[

[18] The method for producing a freeze-dried preparation according to any one of [18].

[0035] [20] The method for producing a freeze-dried preparation according to [19], wherein the polyol mass / alanine mass ratio is from 0.5 to 2.5.

[0036] [21] A method for producing a freeze-dried preparation according to [19] or [20], wherein the antibody mass / alanine mass specific force S is 0.05 to 3.

[0037] [22] The ratio of the mass of the three components of antibody mass, alanine mass, and sucrose mass is 1: 0.3 to 20: 0.

A method for producing a freeze-dried preparation according to any one of [15] to [21], which is 5 to 30.

The invention's effect

[0038] As shown in the Examples, the preparation containing the antibody of the present invention has a polymer, a degradation product, a deamide, an antibody polymer during the storage period even when stored at 25 ° C or 40 ° C for 3 months. It was confirmed that the antibody was stable without increasing its oxidant, and the biological activity of the antibody was retained. The preparation according to the present invention is stable for at least 1 year at low temperature (2 ° C to 8 ° C), and stable for at least 25 months at 3 months and / or at 40 ° C for 1 month.

[0039] 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.

Brief Description of Drawings

[0040] 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 shows remarkable polymer formation, but shows that a formulation combining alanine with trehalose or sucrose is stable.

[Fig. 2] 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 ratio of sucrose in combination with alanine indicates that a formulation with R of 0.25 to 2.5 is stable.

[Fig. 3] Fig. 3 shows changes in the amount of polymer when each lyophilized preparation is stored at 25 ° C or 40 ° C. FIG. The amount of polymer was measured by size exclusion HPLC. This indicates that a preparation with a Q (ratio of antibody mass to alanine mass) of 0.05 to 3 is stable.

[Fig. 4] Fig. 4 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 lyophilized preparations with anti-HLA-DR antibody and anti-CD40 antibody using alanine and sucrose as excipients are stable.

BEST MODE FOR CARRYING OUT THE INVENTION

[0041] Description of Disclosure Related to the Invention

A “stable formulation” does not contain ingredients that are toxic to the patient to whom the formulation is administered, and by adding additives other than active ingredients that do not disrupt the patient's biostasis as much as possible. These active ingredients retain chemical and / or physical and / or biological stability during storage. `` Additives other than active ingredients that do not disrupt the patient's homeostasis as much as possible '' are those whose safety has been sufficiently confirmed by past treatment results, or past administration results have not been! O! / Even if there is toxicity evaluation for cells and animals! /, Means that safety is sufficiently predicted by other methods. “Preserving patient homeostasis” means that additives other than the active ingredient have no biological activity unacceptable to the patient and / or isotonic (essentially with human blood). Have the same osmotic pressure).

[0042] Lyophilization is a lyophilization often used in the preparation of pharmaceuticals!

(Freeze drying) method. 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 component 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 becomes 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 freeze-drying is the moisture content to a level where various molecular events are significantly reduced (this is The product becomes unstable during long-term storage). The lyophilized formulation can also easily withstand the physical stress of transport. The reconstituted product is free of particulate matter and can therefore be administered parenterally, preferably intravenously or intramuscularly or subcutaneously, to a subject without prior filtration.

[0043] 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 enable 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. Longer reversion times degrade product quality because the protein is exposed to longer time in a more concentrated solution. 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.

[0044] Protein stability is measured by various analytical methods. For example, the new 'protein refinement theory and practice', as outlined by RK Scopes, Springer's “Fairark Tokyo Publishing”. Protein stability includes chemical stability, physical stability and biological stability. “Chemical stability” can be assayed by detecting the chemically altered state of the protein. Chemical changes can be assessed by size modification such as clipping, which can be assessed by size exclusion chromatography or SDS-PAGE, charge state changes that can be assessed by ion exchange chromatography (eg, as a result of deamidation), and hydrophobic chromatography. Includes possible changes in the hydrophilic / hydrophobic state (eg resulting from oxidation). “Physical stability” includes the absence of insoluble particulates and / or turbidity and / or agglomeration 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.

[0045] Proteins useful for therapeutic applications include antibodies. Antibodies are expressed on the cell surface. Attempts have been made to use antibodies that bind to proteins and have the effect of inducing cell death or damage to cells for the treatment of cancer and the like. Currently, it is used as a target disease for monoclonal antibodies such as chimeric antibodies (Rituximab) targeting CD20, a receptor on the cell membrane, humanized antibodies targeting Her2 / neu, and malignant tumors. The therapeutic effect is recognized. In addition, as disclosed in the pamphlet of International Publication No. WO2003 / 033538! /, A monoclonal antibody (anti-HLA-DR antibody) against an HLA-DR antibody, which is a type of class II major histocompatibility complex (MHC) molecule Is also considered useful. Antibodies are particularly useful as anti-tumor agents because of their high specificity for antigens with long blood half-lives. 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 cytotoxicity. Attacks against cancer cells of the immune system can be expected by activity (ADCC). In addition, by binding a drug such as a radionuclide or a cytotoxic substance to the antibody, the bound drug can be efficiently delivered to the tumor site. Reducing side effects can be expected by reducing the amount of drug delivered. If a tumor-specific antigen has an activity that induces cell death, administration of an antibody having agonistic activity can be performed. Also, tumor-specific antibody accumulation and tumor growth due to antibody activity Stop or retraction is expected. The antibody is considered to be suitable for application as an antitumor agent due to its characteristics as described above.

[0046] 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, they contain more stable antibodies. There is a need in the art for lyophilized 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 medicinal effect is lyophilized, and the form in which the active ingredient is clearly effective in a solid, It does not contain any additional ingredients that may disrupt biostasis to the patient to whom the product is administered. Here, “the active ingredient shall be clearly effective” means that the active ingredient contained therein maintains its activity and does not lose its pharmaceutical effect. [0048] “Does not disrupt the patient's homeostasis as much as possible! /, An additive other than the active ingredient” refers to those whose safety has been sufficiently confirmed by past treatment results, or no past administration results! It means substances whose safety can be sufficiently predicted by toxicity evaluation for cells, animals 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 are not acceptable to the patient! /, Have no biological activity, and / or are isotonic if possible (human blood And have the same osmotic pressure! /).

[0049] A "stable formulation" is one in which the active ingredient therein retains chemical and / or physical and / or biological stability upon storage. Preferably stable for at least 1 year 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. Stable to freeze-thaw, light irradiation and 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 modifications 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 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. “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. An antibody retains biological stability when it has not undergone chemical or physical changes. Therefore, if the antibody in the preparation retains chemical stability and physical stability, it can be said that the preparation is stable. In other words, whether 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. Stable preparations do not have the ability to increase the amount of antibody polymer, degradation products, deamides, oxidants, etc. during storage so as to reduce the pharmaceutical effect of the preparation, and no insoluble foreign matter or turbidity is observed. The stable formulation of the present invention is Antibodies can be stored for at least 1 year at low temperatures (2 ° C to 8 ° C), or stored for at least 3 months at 25 ° C, or stored at 40 ° C for 1 month The polymers, degradation products, deamides, and oxidants do not increase so as to reduce the pharmaceutical effect of the preparation. Also, even if the product is freeze-thawed or subjected to vibration, the antibody polymer, degradation product, deamide, and oxidant do not increase so as to reduce the pharmaceutical effect of the preparation. The antibody polymer in the stable preparation of the present invention does not increase during storage, for example, when it is measured by size exclusion chromatography after being stored at 25 ° C or 40 ° C for one month, A small proportion of the polymer is desirable. In addition, the degradation product of the antibody in the stable preparation of the present invention does not increase greatly during storage, for example, the degradation product for the antibody in the formulation even if stored for 1 month at 25 ° C or 40 ° C. It is desirable that the ratio of is small. In addition, the amount of deamidated antibody in the stable preparation of the present invention does not increase greatly during storage, for example, even if stored at 25 ° C or 40 ° C for 1 month, It is desirable that the ratio of the deamide 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, even if it is stored at 25 ° C or 40 ° C for 1 month, A small percentage 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 the 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.

[0050] 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. “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.

[0051] The term "antibody" included in this patent is used in the broadest sense, and in particular as long as it retains a monoclonal antibody, a polyclonal antibody, a multispecific antibody, or a desired biological activity. Includes antibody fragments. The antibodies included in this patent are not particularly limited as long as they bind to a desired antigen. Mouse antibodies, rat antibodies, rabbit antibodies, Hedge antibodies, camel antibodies, chicken antibodies, chimeric antibodies, humanized antibodies, human antibodies, etc. Can be used as appropriate. The antibody may be a polyclonal antibody or a monoclonal antibody.

[0052] The present invention relates to a stable lyophilized pharmaceutical preparation containing an antibody. The antibodies in the formulation are prepared using techniques well known to those skilled in the art that are available in the relevant field to produce antibodies. Exemplary monoclonal antibody preparation methods include: (1) purification of biopolymers used as immunogens and / or creation of cells overexpressing antigenic proteins on the cell surface; (2) antigens in animals After immunization by injection into cells, blood is collected and the antibody titer is assayed to determine the timing of removal of the spleen and the like, and then antibody-producing cells are prepared. (3) Myeloma cells ( (4) cell fusion between antibody-producing cells and myeloma, (5) selection of hyperidoma groups producing the desired antibody, (6) splitting into single cell clones (cloning), (7 ) In some cases, culture of high-pridoma to produce a large amount of monoclonal antibody, or breeding of animals transplanted with high-pridoma. (8) In some cases, human monoclonal antibody is obtained from antibody-producing cells such as hyperidoma. The gene to be coded is cloned, incorporated into an appropriate vector, and introduced into a host (eg, mammalian cell line, E. coli, yeast cell, insect cell, plant cell, etc.), and gene recombination technology is used. Preparation of the produced recombinant antibody, (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 also be prepared by methods well known to those skilled in the art.

[0053] The antibody included in this patent has a heavy chain having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequences of the heavy chain and / or light chain of the antibody. And / or an antibody consisting of a light chain is also included. Partial modification (deletion, substitution, insertion, addition) of the amino acid as described above into the amino acid sequence of the antibody of the present invention involves partial modification of the base sequence encoding the amino acid sequence IJ. Can be introduced. This partial modification of the base sequence is a known site-specific mutagenesis method. Can be introduced by a conventional method. (Proc Natl Acad Sci USA., 1984 Vol81: 56 62). 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, OWGoding, Monoclonal Anibodies: rinciples and practices (1993 Academic Press), bacterial toxins such as Pseudomonas aeruginosa toxin, diphtheria toxin, ricin, and methotrexate, such as iodine, yttrium, indium, and technetium. , Mitomycin, calikiamycin and other chemotherapeutic agents (DJKing, Applications and bngineenng of Monoclonal AntiDoaies., 1998 .j .Intenational Ltd .; MLGrossbard., Monoclonal Antibody-Base Therapy of Cancer., 1998 Marcel Dekker Inc), Maytansinoid And other prodrugs (Chari et al., Cancer Res., 1992 Vol. 52: 1 27; Liu et al., Proc. Natl. Acad. Sci. USA, 1996 VoL93: 8681), etc. Those that further enhance the therapeutic effect of diseases such as cancer are also included.

[0054] The antibodies included in this patent also include functional fragments of antibodies. The term “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 ′), Fab ′, Fab, Fv, disulfide binding Fv, single chain Fv (scFv), diapodi, and polymers thereof (DJKing. Applications an a Engineering of Monoclonal Antibodies., 1998 TJ International Ltd) ₀

[0055] The antibody included in this patent is a polyclonal antibody or a monoclonal antibody, and is preferably a human antibody, a humanized antibody or a chimeric antibody. The antibody is preferably IgG, and is preferably any one of IgG subclass IgG1, IgG2, and IgG4. Further, IgG may be an IgG in which amino acid deletion and / or substitution and / or insertion has been performed on a part of the amino acid sequence of the constant region by genetic modification.

[0056] Further, HD3, HD4, HD6, HD7, HD8, HD10, HD4Gl, HD4G2Ser, HD4G4, which are anti-HLA-DR human monoclonal antibodies described in WO 2003/033538, more preferably, HD8G1, HD8GlSer, HD8G2, HD8G2Ser, HD8G4. Of these, the hybridomas producing HD4, HD6, HD8 and HD10 are FERM BP-7771, FERM BP_7772, FERM BP-7773, and FERM BP-7774, respectively. Research Institute Patent Biological Deposit Center (Ibaraki, Japan) It is deposited internationally at Tsukuba Sakai Higashi 1-chome 1 1 Central 6). The hybridomas producing HD3 and HD7 have been deposited internationally as FERM BP-08534 and FERM BP-08536 on October 31, 2003, respectively. Furthermore, this patent also includes a human monoclonal antibody against CD40, preferably KM281-1-10, M281-2-10, which are anti-CD40 antagonist antibodies described in International Publication No. WO2002 / 088186 pamphlet. -1-2, M283-5, KM225-2-56, KM292- 1-24, KM341_6_9, 4D11, 5H10, 11E1, 5G3, 3811, 3411, 3417, F4_465, and KM302_1, M341-, which are anti-CD40 agonist antibodies 1-19, KM643-4-11, 2053, 2105, 3821, 3822, 285, 110, 115, F1-102, F2-103, F5_77, F5-157. Of these, Hypridoma clones KM302_1, KM281_1_10, and M281-2-10-1-2 were dated May 9, 2001 as clones KM341-1-19 as FERM BP_7578, FERM BP-75 79, and FERM BP-7580, respectively. And 4D11 dated September 27, 2001 as FERMBP-7759 and FERMBP-7758, respectively, and clone 2105 dated April 17, 2002 as FERMBP-8024. An international deposit has been made at Tsukuba Sakai Higashi 1-Chome 1-Chome 1-Chuo 6) under the Budapest Treaty. In addition, plasmids with variable regions of F2-103, F5-77 and F5-157 of heavy chain and light chain were ATCC PTA_3302 (F2_103 heavy chain) and ATCC PTA-3 303 (F2) as of April 19, 2001, respectively. -103 light chain), ATCC PTA_3304 (F5_77 heavy chain), ATCC PTA_3305 (F5_77 light chain), A TCC PTC-3306 (F5_157 heavy chain), and ATCC PTA_3307 (F5_157 light chain) as hybridoma clone F1-102 and F4-465 was dated April 24, 2001 as ATCC PTA-3337 and ATCC PTA-3338, respectively (American Type Culuture Collection, 10801 University Blvd., Manassas, Virginia, USA). Virginia 20110-2209 Manassas 10801 (University Benovard) 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.

[0057] The lyophilized preparation according to the present invention can be prepared by preparing a solution preparation containing the above-described antibody and various additives as active ingredients, and then lyophilizing the solution.

[0058] According to a preferred embodiment of the present invention, the lyophilized preparation contains, in addition to the antibody, a polyol, a crystalline substance, and further contains a buffer and a surfactant. [0059] "Freeze-dried", "lyophilized" and "Freeze-dried" means that the substance to be dried is first frozen and then the ice or freezing solvent is placed in a vacuum environment. It means the process of sublimation and removal. 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 process is performed by a known method with a force S.

[0060] The "additive" included in the present invention includes all components included in the lyophilized preparation other than the active component, such as a buffer, a pH adjuster, an isotonic agent, an excipient, and a stabilizer. , Surfactants, preservatives and the like. In addition, one type of additive component that exhibits two or more types of effects is also included.

[0061] "Buffer" refers to an additive that moderates changes in pH by the action of an acid-base conjugate component. The buffer concentration is about 1 mmol / L to about 50 mmo / L, preferably 5 mmol / L to 20 mmol / L, and most preferably about 10 mmol / L, depending on its buffer capacity and / or the desired osmotic pressure. Preferred buffering agents include glutamate (eg, sodium glutamate), citrate and other organic acid buffers. Most preferred is glutamate.

[0062] "Polyol" is a substance having a plurality of hydroxyl groups, and is also used as an excipient in lyophilized preparations. 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). “Reducing sugar” includes a force capable of reducing metal ions, or a hemiacetal group capable of reacting covalently with other amino groups and lysine in protein. “Non-reducing sugar” It does not have these properties of sugar. Examples of reducing sugars are funolectose, mannose, manoleose, ratatose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Examples of non-reducing sugars include sucrose, trenosose, sorbose, meletitose, and raffinose. Examples of sugar alcohols include mannitol, xylitol, erythritol, thritol, sorbitol, and daricerose. Sugar acids include L-darconic acid and its metal salts. The use of reducing sugars in lyophilized formulations is not preferred because it promotes the formation 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 use an amorphous structure during freezing and drying. Non-freezing Illustrative examples of polyols having a crystalline structure include sucrose, trehalose, sorbitol and the like. The polyol of the present invention is preferably a non-reducing sugar and amorphous polyol, or a polyol such as a non-reducing sugar and 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.

[0063] "Crystalline substance" includes crystalline amino acids such as alanine and glycine as long as they take a crystal structure upon freezing. 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.

[0064] Polyols and crystalline substances can act as excipients and tonicity agents. In the present invention, the “excipient” refers to a substance having a function of maintaining the cake shape of a freeze-dried preparation and / or a function of protecting a protein against stress associated with freezing and drying. In addition to polyols and crystalline materials, a common example used for excipients is lactose. An excipient provides useful properties in that it enhances protein stability during long-term storage. These substances can also act as isotonic agents. An “isotonic agent” refers to an agent that adjusts the osmotic pressure so that the subject formulation has essentially the same osmotic pressure as human blood. It is desirable that the isotonic preparation has an osmotic pressure of about 250 to 350 mOsm / kg, and the osmotic pressure ratio is 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.

[0065] In the present invention, a polyol such as sucrose also acts as a stabilizer against antibody aggregation, and plays an important role in reducing the time required for reconstitution of a freeze-dried preparation in a solution without any particulate matter. Also fulfills. 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.

[0066] "Surfactants" include nonionic surfactants such as polysorbates (eg, polysorbate 20, 80, etc.) or poloxamers (eg, poloxamer 188). Added The amount of detergent 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. 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 O. lmg / mL, most preferably about 0.05 mg / mL.

[0067] 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 (ρΗ5 · 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.

[0068] In the present invention, a crystalline substance (most preferably alanine), a non-reducing sugar and an amorphous polyol (most preferably sucrose and trehalose) are added to an antibody as excipients and freeze-dried. A stable lyophilized formulation is provided. In essence, the cake structure is maintained in a crystalline phase composed of a crystalline substance, and frozen and dried by an amorphous phase composed of antibodies and non-reducing sugars and / or amorphous polyols (most preferably sucrose and trehalose). By protecting the protein from moderate stress, a stable lyophilized formulation can be derived.

[0069] The blending ratio and blending amount of the alanine and sucrose excipients used in the present invention are appropriately adjusted according to the antibody concentration which is the active ingredient.

[0070] When R is the mass ratio of sucrose to alanine (sucrose mass / alanine mass) or concentration ratio (sucrose concentration / alanine concentration) present in the lyophilized preparation, stability is good R is 0.25 or more Preferably 0.5 to 2.5, more preferably 1 to 2. Most preferably 1.5.

[0071] Further, when 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, sucrose / Good stability with freeze-dried formulation containing alanine Q between 0.05 and 3, preferably 0

• 05 and 1.5.

[0072] From the combination of R and Q described above, stable lyophilization comprising antibody, alanine and sucrose In the sexing agent, the ratio of the mass of the three components of antibody mass, alanine mass, and sucrose mass is 1: 0.3 to 20: 0.5 to 30. The ratio of the total mass of alanine and sucrose to the antibody is 1: 5 / 6-50.

[0073] The freeze-dried preparation of the present invention is used after being restored.

[0074] "Restoration" is the time required for rehydration of a lyophilized preparation with a solution to obtain a particle-free / clarified solution. An important feature of lyophilized formulations is the product reconstitution time or the time required to rehydrate, requiring 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 with increasing protein concentration. Therefore, longer reversion times degrade product quality because proteins are exposed to more concentrated solutions for longer periods of time. In addition, the user cannot administer until the product is completely rehydrated. This is to ensure complete rehydration to ensure that the product is free of particulates, is administered at the correct dose, and its sterility is not affected. That is, rapid rehydration is advantageous for patients and physicians.

[0075] 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.

[0076] The lyophilized formulation is rehydrated at the time of use with a diluent (eg, water for injection) to give a solution free of particulate matter. The reconstituted antibody solution does not contain particulate matter after long-term storage of the lyophilized cake at ambient temperature. The reconstituted solution is administered parenterally, preferably intravenously, intramuscularly or subcutaneously to the subject.

[0077] The composition of the present invention minimizes the formation of protein aggregates and particulates in an immunoglobulin-containing reagent and ensures that the antibody in solution maintains its immune activity over time. . The composition is a sterile, pharmaceutically acceptable lyophilized solution prepared from an aqueous pre-lyophilized formulation containing antibodies, alanine, and polyol and surfactant in a buffer having a neutral or acidic pH. Contains formulation. The composition of the present invention further comprises alanine and And excipients other than polyols and / or tonicity agents.

[0078] The formulation is administered to mammals, preferably humans, in need of treatment with antibodies, in a known manner, for example, intravenously administered as a bolus or by continuous infusion over time, intramuscular, intraperitoneal, intracerebral, subcutaneous. Administered by intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In a preferred embodiment, the formulation is administered to the mammal by intravenous administration. For such purposes, the formulation is infused, for example, using a syringe or via an intravenous infusion line.

Example

[0079] The present invention is further illustrated by the following examples. The invention will be more 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, IgGl) described in International Publication No. WO2003 / 033538 as an antibody.

[0081] In this example, the formulations shown in Table 1 were prepared, and detailed examination of suitable excipients to be combined with alanine was conducted.

[table 1]

This implementation ί One preparation for 製剤¹ £

[0082] (1) Preparation of preparation specimen

The reagent used in this study was an anti-HLA-DR antibody (approximately 20 mg / mL, Kirin Brewery's Pharmaceutical Production Technology Laboratory (July 2007 1) according to the method described in International Publication No. W02003 / 00 33538 pamphlet. After that, prepared by Kirin Pharma Co., Ltd. Production Technology Laboratory), sodium L-glutamate monohydrate (Japanese Pharmacopoeia Standard), alanine (Ala, Japanese Pharmacopoeia), trehalose (Tor, Japan Pharmacy) ), Sucrose (Suc, Japanese Pharmacopoeia), sodium chloride Lithium (NaCl, Japanese Pharmacopoeia), hydrochloric acid (Japanese Pharmacopoeia), polysorbate 80 (Japanese Pharmacopoeia), and water for injection (Japanese Pharmacopoeia) were used. 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 (manufactured by Pharmacia Biotech). The protein concentration was converted using the OD280 匪 absorbance coefficient ε = 1.4 and adjusted.

[0083] Each preparation specimen was aseptically filtered using a 0.22 m filter (Millipore) in a clean bench, and filled in 5 mL glass vials (conforming to the Japanese Pharmacopoeia). After filling, half-plugging was performed with a rubber plug! / \ Freeze-drying was performed using a freeze dryer manufactured by Nippon Vacuum Technology Co., Ltd. Aseptic dilution of V and solution (placebo) containing anti-HLA-DR antibody for dilution of analysis specimen and analysis blank was performed using 0.22 μm bottle top filter (Nalgen) in a clean bench.

[0084] (2) Test conditions

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

[0085] 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 heat stress.

[0086] (3) Analysis method

Visual appearance: The shape of the cake was examined with the naked eye under a fluorescent lamp, and the appearance was described.

[0087] For the following analysis, after visual inspection, 1 mL of water for injection was added with a syringe and dissolved again.

[0088] Insoluble foreign matter and turbidity: The presence of insoluble foreign matter and turbidity was examined with the naked eye at a position of about 5,000 lux directly under a white light source.

[0089] Size exclusion HPLC assay (SEC): The polymer content and degradation product content were calculated by the size exclusion high-speed liquid chromatography method. Samples were diluted to lmg / mL as necessary and 20 L injections were performed at ambient temperature. For separation, use TSKgel G3000 SWXL 30cm X 7.8mm (manufactured by Tosohichi Co., Ltd.) column, and use 20mmol / L sodium phosphate and 500mmol / L sodium chloride pH 7.0 as mobile phase and analyze at 0.5mL / min flow rate. The time was 30 minutes and detection was performed at 215 nm. The main P The polymer eluted before the catalyst was defined as the polymer, and the polymer eluted after the product was defined as the decomposition product.

[0090] Cation exchange HPLC assay (IEC): Deamide content was calculated by the cation exchange high performance liquid chromatographic method. Sixty specimens diluted appropriately to 5 mg / mL were injected. Detection was performed at 280 nm using TSKgel BIOAssist S (manufactured by Tosoh Corporation) as a separation column. As mobile phase, 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 the analysis was performed using the optimum gradient conditions. Degraded substances that eluted ahead of the main peak were defined as deamides.

[0091] Hydrophobic HPLC assay (HIC): The content of oxidant was calculated by hydrophobic chromatography. Twenty samples, each appropriately diluted to 1 mg / mL, were injected using a sample processing solution (20 mmol / L sodium phosphate, 0.984 mol / L ammonium sulfate, 7.5 mmol / L methanoline). Analysis and detection were performed at 215 nm using TS gel Buty NPR (manufactured by Tosoh Corporation) as a separation column. The mobile phase is 20 mmol / L sodium phosphate, ρΗ6 · 5 as A, and 20 mmol / L sodium phosphate, 1.2 mol / L ammonium sulfate, ρΗ6.5 as Β, and the analysis is performed using the optimal gradient conditions. I went. In addition, the deteriorated substance eluted ahead of the main peak was defined as an oxidant.

[0092] Reduction SDS-PAGE assay: The sample solution was diluted to 400 g / mL as necessary. ¾DS Sample Buffer (Invitrogen), sample Reducing Agent (Invitrogen¾¾), 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 (manufactured by Invitrogen) for electrophoresis. Apply 5 L 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. went. The gel after swimming 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 Da proteins) are run simultaneously.

[0093] Non-reducing SDS-PAGE assay: The sample solution was diluted to 400 μg / mL as necessary. LDS Sample Buffer (Invitrogen) and H0 were added to the sample solution to obtain a non-reduced sample solution. The electrophoresis gel was filled with Tris-Acetate SDS Running buffer (Invitrogen) for electrophoresis. Apply 5 L of the sample solution to 3-8% Tris_Acetate Gel (Invitrogen), perform electrophoresis at a constant voltage of about 125 V, and the blue color of bromophenol blue in the sample solution is near the bottom of the gel. Went to move on. 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 product, molecular weight markers (including 220 KDa, 116 KDa, 97.4 Da, 63.3 KDa, 54.4 Da, and 36.5 KDa proteins) are run simultaneously.

[0094] Osmotic pressure ratio: V was measured using an automatic osmotic pressure measuring apparatus (OSMO STATION OM-6050 manufactured by ARKRAY, Inc.), and the ratio of the measured physiological saline to the osmotic pressure was calculated. pH: pH was measured using an automatic pH measuring device (Metler 'MP-230 manufactured by Toledo Co., Ltd.). Measurement was performed after calibration using standard solutions of pH 4, pH 7, and pH 9 at the start of measurement.

[0095] Moisture measurement: The moisture content was measured using a trace moisture analyzer (AQ-2000, Hiranuma Sangyo Co., Ltd.) connected to an automatic moisture vaporizer (LE-20SA, Hiranuma Sangyo Co., Ltd.), and the moisture content was calculated.

[0096] (4) Results and discussion

Figure 1 shows the change in polymer when each lyophilized formulation was stored at 25 ° C and 40 ° C. The amount of polymer was measured by size exclusion chromatography. In the formulation 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 formulation using alanine and sodium chloride as excipient, foreign matter and turbidity were confirmed by visual inspection with the naked eye after redissolving, and the increase in polymer was also largely unstable. The formulation 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 the polymer was dramatically suppressed by stringing polanolin (trehalose, sucrose) used above with alanine, indicating a high stability. In addition, in other analysis items (cake shape, foreign matter, turbidity, decomposed product, insoluble fine particles, amide, and oxidant), a combination of alanine and polyol shows almost no change before and after thermal stress loading. It was stable.

[0097] 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. It is desirable to use an amorphous polyol, more preferably a non-reducing and amorphous polyol, and most preferably, trehalose or sucrose as the polyol.

[0098] Example 2 Sucrose-alanin ratio

In Example 1, it was found that trehalose or sucrose is most preferable as the polyol combined with alanine. In this example, sucrose was used as the polyol, the concentration of alanine was kept constant, the concentration of sucrose mixed with alanine was changed, and the optimum concentration ratio R for polymer suppression was examined. Here, R represents the mass of sucrose / alanine present in the lyophilized product.

[0100] A freeze-dried preparation was prepared using an antibody against HLA-DR (anti-HLA-DR antibody, IgGl) described in International Publication No. WO2003 / 033538 as an antibody.

[0101] In this example, the preparations shown in Table 2 were prepared, and a detailed examination of an appropriate concentration of sucrose to be combined with alanine was conducted.

[Table 2]

Formulation used in this example -ΪΕ

[0102] (1) Materials and methods

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

[0103] (2) Test conditions

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

[0104] (3) Results and discussion

Figure 2 shows the change in polymer when each lyophilized formulation was stored at 25 ° C and 40 ° C. The amount of polymer was measured by size exclusion chromatography. It was clarified that the polymer formation was suppressed as the mass of sucrose mixed increased, indicating that it 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 that obtained by the size exclusion chromatograph was obtained.

[0105] A cake is formed at any of the concentrations studied in this example, particularly R = l. A beautiful cake was formed at 5 or less and showed a shape that could withstand long-term storage. The 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.

[0106] In this example, the optimum sucrose mass ratio R (sucrose mass / alanine mass) to be combined with alanine as an excipient of a freeze-dried preparation for stabilizing the antibody was revealed. R having good stability was 0.25 or more, preferably 0.5 to 2.5, more preferably 1 to 2, and still more preferably 0.75 to 2. Most preferably it was 1.5

[0107] Example 3 Effect of antibody concentration

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

[0108] In this example, the preparations shown in Table 3 were prepared, and the sucrose / alanine formulation (R = 1.5) optimal for stabilizing the antibody obtained in Example 2 was given to the preparations having different antibody concentrations. The impact was evaluated. Where Q is the ratio of antibody mass to alanine mass. That is, Q represents the antibody mass / alanine mass in the lyophilized preparation.

[Table 3] Formulations used in this project-One W

[0109] (1) Materials and methods

The materials, materials and analysis methods used in this example are the same as those shown in Example 1.

[0110] (2) Test conditions

Similarly to the method shown in Example 1, the samples were stored in an incubator (manufactured by TAB AI 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.

[0111] (3) Results and discussion Figure 3 shows the polymer changes when each lyophilized formulation was stored at 25 ° C and 40 ° C. The amount of polymer was measured by size exclusion chromatography. The formulation with the highest antibody concentration (the highest amount of antibody) Q = 3 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 antibody concentration in Example 1 was 10 mg / mL and the formulation of alanine alone was about 9% when stored at 40 ° C for 3 months, the polymer formation was sufficiently suppressed by V, Obviously. The formulation with the lowest antibody concentration (the least antibody amount! /,) Q = 0.05 was stable with almost no change observed before and after stress loading. In other analysis items (foreign substances, turbidity, decomposition products, deamides, and oxidants), almost no change was observed before and after stress loading, and the analysis was stable.

[0112] 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 for Q = 0.05 to 3. That is, a stable freeze-dried preparation is obtained with a freeze-dried 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 antibody mass (antibody mass is 1), it is 1: 5 / 6-50, preferably 1:;!-20, more preferably 1:;! To 10, particularly preferably 1: 5.

[0113] Example 4 Investigation with other antibodies

In this study, an optimal alanine / sucrose formulation (R = 1.5) for stabilizing the antibody obtained in Example 2 was used for recombinant complete HgG4 against CD40 published in International Publication No. WO0563981. The stability of the preparation was examined using antibody 4D11 (anti-CD40 antibody).

[0114] In this example, the preparations shown in Table 4 were prepared, and the effect of the type of antibody on the stability of the preparation was evaluated.

[Table 4]

Formulations that have fallen into this implementation

Active ingredient 几几体 ¾ 赋 Surfactant 铜 pH

1 0.05rng / m

Anti-HLA-DR antibody 20mg / mL 30mg / m

10 mg / mL alanine polysorbate 5.5

2 Scroll

Anti-CD40 antibody 80 [0115] (1) Materials and methods

The materials, 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.

[0116] (2) Test conditions

[0117] (3) Results and discussion

Figure 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, with the value before lyophilization being 1). It was. The amount of polymer was measured by size exclusion HPLC. Also in the anti-CD40 antibody, in the preparation using alanine and sucrose as excipients, suppression of polymer formation during the storage period was confirmed, and the antibody in this preparation was stably maintained regardless of temperature and time. In addition, the other analysis items (foreign matter, turbidity, degradation products, deamides, and oxidants) were stable with almost no change observed before and after thermal stress loading.

[0118] In this example, it was found that a stable lyophilized preparation using alanine and sucrose as excipients is stable regardless of the type of antibody.

[0119] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

The scope of the claims

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

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

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

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

[5] The mass of alanine and polyol with respect to antibody mass of 1 is 5/6 to 50;

6. The freeze-dried preparation according to claim 5, wherein the specific mass of polyol / alanine mass is 0.5 to 2.5.

Yes

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

[8] The ratio of the three masses of antibody mass, alanine mass, and sucrose mass is 1: 0 · 3 to 20: 0 · 5 to 3

The lyophilized preparation according to any one of claims 1 to 7, which is 0.

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

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

[I I] The freeze-dried preparation according to claim 9 or 10, wherein the concentration of the buffer is 10 mM.

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

[13] The freeze-dried preparation according to claim 12, wherein the surfactant is polysorbate.

[14] The freeze-dried preparation according to any one of claims;! To 13, which is a pharmaceutical preparation containing an antibody as an active ingredient.

[15] A method for producing a stable freeze-dried preparation comprising adding alanine and polyol to an antibody and freeze-drying.

16. The method for producing a freeze-dried preparation according to claim 15, wherein the polyol is a non-reducing sugar and is an amorphous polyol.

17. The method for producing a lyophilized product according to claim 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 claim 17, wherein the polyol is sucrose or trehalose.

[19] The mass of alanine and polyol with respect to antibody mass 1 is 5/6 to 50.

19. A method for producing a freeze-dried preparation according to any one of 18 above.

20. The method for producing a lyophilized product according to claim 19, wherein the specific mass of polyol / alanine mass is 0.5 to 2.5.

21. The method for producing a freeze-dried preparation according to claim 19 or 20, wherein the antibody mass / alanine mass ratio is 0.05 to 3.

[22] The ratio of the mass of antibody, alanine, and sucrose is 1: 0.3 to 20: 0.5 to 3

The method for producing a freeze-dried preparation according to any one of claims 15 to 21, which is 0.