JP2005060378A - Freeze-dried preparation containing physiologically active protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an improvement in production of stable freeze-dried preparation containing a physiologically active protein, such as IL (interleukin)-11, without having such anxious matters that the freeze-dried preparation containing the physiologically active protein becomes cloudy when redissolved and containers are broken in the production. <P>SOLUTION: A method for preventing the freeze-dried preparation containing the physiologically active protein from becoming cloudy when redissolved comprises accelerating crystallization of a buffering agent in a freezing process for dealing with various problems of the freeze-dried preparation containing the physiologically active protein, especially, a problem of becoming cloudy when redissolved, namely, a problem that solubility (turbidity) of the preparation when reconstructed is controlled by not only a structural change in a solution after dissolved but also physical properties of a preparation solid (freeze-dried cake). The method solves the problem that the preparation becomes cloudy when redissolved, is effective for stabilizing the preparation, and further solves the problem that the containers are broken in the production. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a method for preventing turbidity of a freeze-dried preparation containing a physiologically active protein, a method for avoiding damage to an ampule, a lyophilized preparation containing a physiologically active protein in which prevention of white turbidity during re-dissolution (avoidance of ampule damage) is achieved, and a method for producing the same. More specifically, the present invention relates to the above improvement of a basic protein such as interleukin-11 (hereinafter referred to as IL-11).

Bioactive proteins are used in various fields as pharmaceuticals, and include various interferons, hematopoietic factors, TPA, urokinase, various CSFs, and various interleukins (IL). When these are administered parenterally, the preparation is generally liquefied at the time of administration. Physiologically active proteins have poor storage stability, and there are many difficulties in preparing liquid formulations. In general, the prepared preparation is lyophilized, liquefied with a solution at the time of use, and administered parenterally.
In lyophilized preparations, various stabilizers have been studied, and a combination of useful proteins and surfactants (Patent Document 2), mixing of IL-12 and surfactants (Patent Document 3), non-use of IL-6 There are reports of addition of reducing sugar (Patent Document 4), addition of various sugars to G-CSF (Patent Document 5), addition of a water-soluble heterocyclic compound to human growth hormone (Patent Document 6), and the like. Patent Documents 2, 3, and 4 have a problem of avoiding a decrease in the activity of the proteins described therein, and Patent Documents 5 and 6 have a problem of avoiding irreversible aggregate formation.

  IL-11, a physiologically active protein, is a protein that is naturally derived or produced by recombinant gene manipulation methods, and stimulates various hematopoiesis and immune functions. Genetics Institute (currently Wyeth) developed "Neumega (product name)" consisting of recombinant human IL-11 (rhIL-11), an N-terminal proline deletion, For the prevention of severe thrombocytopenia and improvement in avoidance of platelet transfusion seen after bone marrow prevention chemotherapy in Japan. This preparation is a freeze-dried preparation described in Patent Document 1, and contains IL-11 as a concentration after re-dissolution of the preparation, 5 mg / ml, 10 mM sodium phosphate (pH 7.0), and 300 mM glycine.

International Publication No. WO95 / 28951 (corresponding US Pat. No. 6,270,757) JP 2001-192343 A JP 2002-275197 A JP-T 8-502722 Publication JP-T 8-504784 Japanese Patent Laid-Open No. 10-265404

An object of the present invention is to provide a physiologically active protein, preferably IL-11, which is free from concerns such as prevention of white turbidity at the time of re-dissolution of a lyophilized preparation containing a physiologically active protein, good re-solubility and breakage of a container at the time of production. It is to provide an improvement in the production of a stable lyophilized formulation comprising.
Since IL-11 is relatively stable in a pH range near neutral in an aqueous solution, it is desirable to formulate it in the form of a liquid preparation for injection that is conventionally advantageous in various ways. However, since it hydrolyzes depending on the temperature, it is difficult to store the liquid preparation for a long period of time, which is practically required. In practice, it must be a lyophilized preparation. In order to obtain a lyophilized preparation, the IL-11 solution is rapidly cooled to -30 ° C. or lower, frozen by breaking the supercooling of the solution, and then dried. A cloudiness was observed.
In general, some lyophilized preparations containing physiologically active proteins are observed to be slightly soluble and cloudy in part of the lyophilized cake upon re-dissolution. The lyophilized preparation of bioactive protein is prepared by re-dissolving with water for injection during clinical use. At that time, it is quickly re-dissolved so that the presence or absence of abnormalities in the preparation, such as contamination with foreign substances, can be easily confirmed. It is necessary to A freeze-dried preparation that causes white turbidity upon re-dissolution is considered difficult to handle in clinical settings, and a method for preventing such white turbidity upon re-dissolution has been desired.
Furthermore, when manufacturing a freeze-dried preparation containing a physiologically active protein, a container such as an ampoule is frequently damaged. Foreign materials should not be mixed in the injection, but small container breakage during production may cause debris into many other non-damaged products due to scattering, which greatly affects the quality of the entire product and production cost. give.

The present inventor conducted various studies to solve the problems such as the above-mentioned transient cloudiness, analyzed the protein interaction in the detailed lyophilization process, and as a result, various lyophilized preparations containing physiologically active protein were analyzed. Among the problems, the problem of white turbidity at the time of re-dissolution, that is, the solubility (turbidity) at the time of reconstitution dominates not only the structural change in the solution after dissolution but also the preparation solid (lyophilized cake) It was found that this is a solid physical property derived from the physical properties of the lyophilized cake, ie, the distribution and state of proteins and buffers in the freeze-dried cake. If the crystallization of the buffering agent is promoted during the freezing process, the problem of cloudiness at the time of re-dissolution of the physiologically active protein-containing lyophilized preparation is solved, the solubility is improved, and the stability of the preparation is effective. The present invention has been completed by finding out that the manufacturing container breakage problem and the like can be solved.
That is, this invention consists of the following.
1. A method for preventing white turbidity at the time of re-dissolution of a lyophilized preparation, comprising means for crystallizing a buffer in a freezing step of a lyophilized preparation containing a physiologically active protein containing a physiologically active protein and a buffer.
2. The method for preventing white turbidity at the time of redissolving, wherein the means for crystallizing the buffering agent performs pretreatment under a temperature condition of −20 ° C. to 0 ° C. before freeze-drying treatment.
3. The method for preventing white turbidity at the time of re-dissolution of the lyophilized preparation according to 1 or 2 above, wherein the physiologically active protein is basic.
4. The method for preventing white turbidity at the time of re-dissolution of the freeze-dried preparation according to 3 above, wherein the physiologically active protein is interleukin 11.
5. The method for preventing white turbidity at the time of re-dissolution of the lyophilized preparation according to the above 1 to 4, wherein the buffer is sodium phosphate and / or histidine.
6. A method for avoiding breakage of ampules in a freeze-dried preparation, comprising means for crystallizing a buffer in the production process of a freeze-dried preparation containing a bioactive protein containing a bioactive protein and a buffer.
7. The ampoule breakage avoiding method as described in 6 above, wherein the means for crystallizing the buffering agent performs pretreatment under a temperature condition of −20 ° C. to 0 ° C. before the freeze-drying treatment.
8. The ampoule breakage avoiding method of the freeze-dried preparation according to 6 or 7, wherein the physiologically active protein is basic.
9. The ampoule breakage avoiding method of the freeze-dried preparation according to the above 8, wherein the physiologically active protein is interleukin 11.
10. A method for producing a lyophilized preparation containing a physiologically active protein comprising the method according to any one of 1 to 10 above.
11. A lyophilized preparation containing a physiologically active protein prepared by the production method described in 10 above.

The present invention relates to a method for preventing turbidity at the time of re-dissolution of a freeze-dried preparation and an ampoule characterized by promoting crystallization of the buffer in a freezing step of a freeze-dried preparation containing a physiologically active protein containing at least a physiologically active protein and a buffer. This is a damage avoidance method.
Here, the physiologically active protein is a protein used as a pharmaceutically active ingredient, and specifically includes cytokines, growth factors, hematopoietic factors, peptide hormones, enzymes and the like. Specifically, cytokines include interferons (α type, β type, γ type, etc.), interleukins (IL-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23), tumor necrosis factor (TNF), and other growth factors include bone morphogenetic factors (BMP-1, 2, 3, 4), nerve growth factor (NGF-1, 2 etc.), neurotrophic factor (NTF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1, 2, 3 etc.), fibroblast proliferation Factors (aFGF, bFGF), platelet-derived growth factor (PDGF), cartilage-derived factor (CDF), transforming growth factors (TGF-α, -β, etc.) and the like.
Hematopoietic factors include colony stimulating factors (G-, M-, GM-CSF, etc.), thrombopoietin (TPO), erythropoietin (EPO), platelet growth stimulating factor, etc., peptide hormones include growth hormone (GH), Growth hormone releasing factor (GRF), corticotropin (ACTH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), melanocyte stimulating hormone (MSH) ), Insulin, glucagon, prolactin, calcitonin, natriuretic peptide, etc. Enzymes include tissue plasminogen activator (tPA), urokinase (UK), superoxide dismutase (SOD), asparaginase, kallikrein, streptokinase, protein C , Metalloproteases, factor VIII and IX, pull An oxidase etc. are mentioned.
Others include cytochrome C, phospholipase A2, lysozyme, acylphosphatase, elastase, galactose oxidase, NGF, trypsin, Kunitz inhibitor, lactoferrin, ribonuclease A, blood coagulation factor XI, plasmin, plasminogen proactivator, adenyl sankinase , Avidin, alcohol dehydrogenase, proelastase, enterotoxin B, enterotoxin C1, kallikrein, chymotrypsin Aα, chymopapain A, chymopapain B, D-glyceraldehyde phosphate dehydrogenase, glucose 6 phosphate isolase, glutaminyl tRNA cyclotransferase, Keratinase, TSH, highly charged iron sulfate protein, colicin E1, subtilisin, cephalosporinase, DNA polymerase β, trypsinogen , Troponin-inhibiting subunits, nucleases, bacteriocins DFB, papain, ficin, plasma B2 and the like.

Preferred physiologically active proteins of the present invention are basic proteins, specifically lactoferrin, ribonuclease A, blood coagulation factor XI, plasmin, plasminogen proactivator, adenyl sankinase, avidin, alcohol dehydrogenase, pro Elastase, Enterotoxin B, Enterotoxin C1, Kallikrein, Chymotrypsin Aα, Chymopapain A, Chymopapain B, D-Glyceraldehyde phosphate dehydrogenase, Glucagon, Glucose 6-phosphate isolase, Glutaminyl tRNA cyclotransferase, Keratinase, TSH, High electric iron Sulfate protein, colicin E1, subtilisin, cephalis polynase, DNA polymerase β, trypsinogen, troponin inhibitor subunit, nuclease, bacteriocin DFB, papain, ficin, Razma B2, cytochrome C, phospholipase A2, lysozyme, acyl phosphatase, elastase, galactose oxidase, NGF, trypsin and Kunitz inhibitor, etc., cytochrome C, phospholipase A2, lysozyme, acyl phosphatase, elastase, galactose oxidase, NGF, trypsin, More preferred are Kunitz inhibitors and interleukins. Of these, IL-11 is particularly preferred. The basic protein referred to in the present invention means a protein having an isoelectric point greater than 7, and the isoelectric point can be easily measured by isoelectric focusing or the like. .
The physiologically active protein in the present invention has a molecular weight of about 1,000 to 200,000, preferably about 5,000 to 70,000. These physiologically active proteins may be naturally derived or modified by gene recombination techniques, or may be modified (for example, chemically modified by polyethylene glycol or the like). These may be used as monomers or as homo or hetero multimers.

The physiologically active protein selected as the most suitable embodiment of the present invention is IL-11, and examples thereof include proteins described in US Pat. No. 5,215,895, US Pat. No. 5,270,181, and US Pat. No. 5,292,646. Also included are proteins produced by recombinant gene manipulation methods, proteins purified from cell sources producing IL-11, proteins synthesized by chemical methods, or proteins obtained by combinations of the above, and in particular N-terminal proline Recombinant human IL-11 (rhIL-11), which is a deletion, can be selected as suitable. Any other IL-11 can be selected as long as the object of the present invention can be achieved.
In this specification, IL-11 includes not only natural IL-11 but also the sequence of natural IL-11 or a sequence in which several amino acid sequences are substituted, deleted and / or inserted. Means a protein exhibiting IL-11 activity (hematopoietic action).

  In the present invention, the concentration of the physiologically active protein before lyophilization is preferably adjusted to a concentration of 0.1 to 20 mg / ml, more preferably 1 to 10 mg / ml, more preferably 3 to 8 mg / ml. Adjusted. If desired, a stabilizer or / and a solubilizing agent for each protein can be selected and added within a range to achieve each target effect. For example, in the case of IL-11, the protein concentration at the time of preparation prior to lyophilization is preferably in the range of 0.1 mg / ml to 20.0 mg / ml, more preferably 1 mg / ml to 10 mg / ml, most preferably about 5 mg / ml. It is. An amino acid, preferably glycine, is added as a solubilizing agent, and the optimum concentration is preferably in the range of 100 mM to 400 mM, more preferably 150 mM to 350 mM, and most preferably about 300 mM.

In the present invention, the buffer means an additive or the like for the purpose of stabilizing the pH of an aqueous solution, and those commonly used in the field of pharmaceutical production can be selected.
In the case of formulation of IL-11, it is possible to select a phosphate buffer containing sodium phosphate. As other buffering agents, histidine, Tris buffer, hepes buffer, and the like can be selected. Further, for example, sodium phosphate and histidine can be used in combination.
The concentration of a suitable buffer at the time of preparation before lyophilization is in the range of 5 mM to 40 mM, more preferably 7 to 30 mM, particularly preferably about 10 to 20 mM. When IL-11 is selected and sodium phosphate is used, it is in the range of 5 mM to 40 mM, preferably 10 mM, and in the case of histidine, it is in the range of 5 mM to 40 mM, preferably about 20 mM.

The present invention mainly uses accelerating crystallization of the buffer in the freezing step. In normal freeze-drying, a drug solution-filled product is stored in a freeze-drying chamber and left on a shelf. Next, the shelf of the freeze-drying cabinet is cooled to −30 ° C. or lower to freeze the filled product. After freezing, the inside of the freeze-drying chamber is depressurized, and the shelf temperature is raised to a temperature at which the filled product does not melt to sublimate the water to perform primary drying. Then, secondary drying is performed by raising shelf temperature and removing adhering water.
In such a normal lyophilization process, means selected from the following procedures can be specifically introduced in the present invention in order to promote crystallization of the buffer.
1) Pre-treatment is performed at a temperature of -20 ° C to 0 ° C before freeze-drying.
2) Freeze the solution at -20 to 0 ° C by reducing the pressure.
Further, in addition to the above-mentioned means for promoting crystallization of the buffer which is the method of the present invention, by combining at least one selected from the following means, it is possible to achieve white turbidity prevention upon re-dissolution of the lyophilized preparation.
3) Add a nonionic surfactant to the adjustment solution.
4) Add sugars to the adjustment solution.

  Freeze-dried preparations made by conventional freeze-drying methods, especially transient turbidity that occurs when redissolving the basic protein IL-11, is probably due to the transient conformation of the bioactive protein that occurs during re-dissolution. Due to the change, especially when the bioactive basic protein and the amorphous buffer coexist in close proximity in the preparation (in the freeze-dried cake) (when manufactured by the conventional freezing method), the protein surface One factor is considered to be temporarily hydrophobic. In the present invention, it has been found that in order to eliminate the influence of the buffering agent, the freezing step may be completed while the buffering agent is crystallized. In order to crystallize the buffering agent, processing temperature, processing time, processing pressure, introduction of a stabilizer and the like can be appropriately selected.

  The pretreatment under the temperature condition of −20 ° C. to 0 ° C. before the drying treatment is an example of specific means for crystallizing the buffer agent. It is possible to select a temperature condition of -15 ° C to -2 ° C, more preferably -10 ° C to -2 ° C for pretreatment. A solution (preparation solution) containing a physiologically active protein is forcibly frozen as a pretreatment at a temperature of −20 ° C. or higher, and the buffer present in the preparation solution is crystallized in advance by this pretreatment. By this treatment, the interaction between the physiologically active substance and the buffering agent is avoided, and then freeze drying is performed after cooling from −20 ° C. to −50 ° C., which is a normal freeze drying treatment.

  When the liquid filtered through a filter with a very small pore size (0.22 μm) like an injection is added to a clean container (ampoule or vial) and frozen, there are very few substances that become the core of ice. Normally, it does not freeze until it reaches a low temperature of around 20 ° C or lower. Therefore, in order to freeze at high temperature, ice crystal nuclei are added, specifically, the freeze-drying chamber is cooled in advance from −30 ° C. to −50 ° C. to form ice nuclei in the freeze-drying chamber, Alternatively, it is also effective to use a means for reducing the pressure of the solution (approximately 10 mmHg or less), irritating the surface of the solution and freezing, and can be used in the present invention.

  When the usual rapid cooling process as described above is employed in the combination of IL-11 and phosphate buffer, the solution is supercooled above -20 ° C and then the whole solution is frozen at once. In this case, a phosphate buffer such as sodium phosphate cannot be crystallized and is frozen in an amorphous state. Such an amorphous phosphate ion electrostatically interacts with a physiologically active basic protein, and thus causes transient cloudiness upon re-dissolution. Therefore, by selectively crystallizing sodium phosphate by changing the freezing temperature from 0 ° C. to −20 ° C., and avoiding electrostatic interaction, a freeze-dried product with good re-dissolution and white turbidity prevention can be obtained. It is thought that it can be obtained. At such a high temperature (0 ° C. to −20 ° C.), even when freezing is started, the entire solution is not completely frozen, and an unfrozen liquid remains, so that molecular mobility sufficient to crystallize sodium phosphate. Is held. In practice, it is more preferable to freeze in the range of −2 ° C. to −15 ° C., and in order to completely crystallize sodium phosphate, it is desirable to leave it in this temperature range for several hours or more.

When pretreatment is not applied under the temperature condition of −20 ° C. to 0 ° C. during freezing (in the case of conventional freezing), sodium phosphate is frozen in an amorphous state, but is crystallized after the drying process. To do. In the crystallization in the drying step, the ampoule is damaged because the volume of the crystallization expands with the crystallization of water. By adding the pretreatment, the sodium phosphate and water are already crystallized before entering the drying process, so that the volume expansion in the drying process does not occur and the ampoule is not damaged. In this way, the pretreatment not only prevents white turbidity but also avoids damage to the ampoule.

  Conventionally, it has been reported that in the case of a protein preparation, insolubilization at the time of re-dissolution becomes a problem in quality. However, in order to solve this problem, as reported by Sarciaux et al. In Journal of Pharmaceutical Science Vol. 88, No. 12: 1354-1361 (1999), There are known techniques such as adding various additives to bovine IgG, or improving the dissolution at the time of re-dissolution by adding annealing during freezing. In general, annealing is a process in which a sample completely frozen at -30 ° C or lower is left at a high temperature of -30 ° C or higher (less than 0 ° C) for several hours, cooled again to -30 ° C or lower and dried. It is performed for the purpose of stabilizing the drug, shortening the drying time, and preventing the freeze-dried cake from collapsing. The present invention is essentially different from this so-called annealing, in which a protein preparation is frozen by introducing a pretreatment at a temperature of -20 ° C or higher, and is rapidly frozen at a low temperature of -30 ° C or lower. It is essentially different from things. In addition, it has been reported that insoluble aggregates of bovine IgG have been reduced by surfactants such as polysorbate 80, but they have not disappeared. It differs from the technology that achieved solubility.

  In the case where the pretreatment is performed at a temperature of -20 ° C to 0 ° C, the pretreatment may be performed under reduced pressure conditions (10 mmHg or less, preferably 1 mmHg or less). By pre-processing with reduced pressure, if the set temperature is set to 0 ° C., the actual product temperature can be stopped at about −10 ° C., and a sufficient target effect can be achieved.

  The processing time of the pretreatment in the above-mentioned “Pretreatment under the temperature condition of −20 ° C. to 0 ° C. before the freeze drying treatment” and “Pretreatment under the reduced pressure condition before the freeze drying treatment” is as follows. Generally, it is 10 minutes or more, preferably 5 to 24 hours, more preferably 10 to 20 hours.

  The present invention provides means for preventing white turbidity during remelting and means for avoiding ampoule breakage during the drying process by selecting the above-described procedure.

  Thus, the parenteral pharmaceutical preparation produced by introducing the method for preventing white turbidity at the time of re-dissolution of the lyophilized preparation is not particularly limited as long as it is usually a pharmaceutically acceptable dosage form. In addition, the lyophilization conditions at the time of manufacturing the lyophilized preparation can be appropriately set as known per se, except for the conditions related to the pretreatment conditions.

  As a method for producing a lyophilized preparation containing a physiologically active protein of the present invention, a method represented by an example in which IL-11 is selected as the physiologically active protein shown below can be employed. First, an aqueous solution containing IL-11 and a phosphate buffer are mixed to obtain a desired concentration to prepare a solution. The prepared liquid is filled in a container, and the shelf in which the ice nuclei are formed in the freeze-drying chamber by cooling the freeze-drying chamber from -30 ° C to -50 ° C in advance is heated to -15 ° C and filled with the solution. Receive the container. After being left overnight, it is cooled to -20 ° C. or lower, completely frozen, and then dried under reduced pressure to obtain the lyophilized preparation of the present invention.

  In the lyophilized preparation containing the physiologically active protein of the present invention, pharmaceutical additives that are usually added to parenteral pharmaceutical compositions (for example, solubilizers, preservatives, stabilizers, emulsifiers, soothing agents, isotonic agents) , Buffers, excipients, colorants, thickeners). For example, L-arginine, cyclodextrins, etc. are mentioned as a solubilizing agent. Examples of the preservative include sodium benzoate and methyl paraoxybenzoate. Examples of the emulsifier include lecithin. Examples of soothing agents include benzyl alcohol and chlorobutanol. Examples of isotonic agents include sodium chloride. Examples of the excipient include maltose. Examples of the thickener include hyaluronic acid.

  The present invention provides a method for producing a lyophilized preparation containing a physiologically active protein having good resolubility upon re-dissolution of the lyophilized preparation without breakage of the container. Achieved improvement. The parenteral pharmaceutical composition or preparation comprising the lyophilized preparation containing the physiologically active protein of the present invention exhibits excellent storage stability in a solution state or a lyophilized state. In particular, in the freeze-dried state, the heat stability is good, and the freeze-dried preparation further exhibits excellent re-solubility and white turbidity prevention effect upon re-dissolution.

  Hereinafter, the present invention will be described with reference to Examples, Reference Examples, Experimental Examples, and the like, but the present invention is not limited thereto. In addition, although IL-11 (rhIL-11) was used as a specific example, each method known per se is similarly applied to other proteins. Examination of pretreatment conditions for freeze-drying treatment was conducted in Examples 1 to 5, Experimental Examples, and Reference Examples 1 to 3 below. In addition, a reference example means the preparation method of the sample for using for a comparative experiment.

(Reference Example 1)
Preparation of freeze-dried preparation by rapid cooling (Sample 1)
Sodium phosphate was dissolved so that IL-11 was contained at 5 mg / ml, glycine at 300 mM, and phosphoric acid at 10 mM, the pH was adjusted to 7, and a container (glass ampule) was filled. The lyophilized preparation was prepared by placing this in a freeze-dried container that had been cooled to -20 ° C or lower, rapidly cooling to -20 ° C or lower, and then performing decompression and drying.

(Reference Example 2)
Preparation of freeze-dried preparation by slow cooling (Sample 2)
Sodium phosphate was dissolved so that IL-11 contained 5 mg / ml, glycine 300 mM, and phosphoric acid 10 mM, the pH was adjusted to 7, and the container (glass ampule) was filled. Put this in a freeze-drying chamber, lower the temperature at a rate of about 10 ° C per hour, cool to -20 ° C or less, and then enter a container filled with the same liquid as a reference for further turbidity. After completely freezing, a freeze-dried preparation was prepared by reducing the pressure and drying.

(Reference Example 3)
Freeze-dried preparation prepared by heating after cooling (annealing) and cooling again (sample 3)
Sodium phosphate was dissolved so that IL-11 contained 5 mg / ml, glycine 300 mM, and phosphoric acid 10 mM, the pH was adjusted to 7, and the container (glass ampule) was filled. Place this in a freeze-drying chamber, cool it to -20 ° C or lower, raise the temperature to about -6 ° C, hold it for several hours, cool it again to -20 ° C or lower, and then add a turbid container filled with the same liquid as a control. A lyophilized preparation was prepared by warehousing and completely freezing them, followed by decompression and drying.

Freeze-dried preparation pretreated at -15 ° C
Dissolve sodium phosphate so that it contains IL-11 5 mg / ml, glycine 300 mM, and phosphoric acid 10 mM, adjust the pH to 7 and fill the container (glass ampule). The shelf in which ice nuclei were formed in the freeze-drying chamber after cooling to −50 ° C. was heated to −15 ° C., and the container filled with the solution was received. After standing overnight, the mixture was cooled to −20 ° C. or lower, and further, as a turbidity control, a container filled with the same liquid was received and completely frozen, and then a freeze-dried preparation was prepared by a method of reducing pressure and drying.

Freeze-dried preparation pretreated at -10 ° C
Dissolve sodium phosphate so that it contains IL-11 5 mg / ml, glycine 300 mM, and phosphoric acid 10 mM, adjust the pH to 7 and fill the container (glass ampule). The shelf on which ice nuclei were formed in the freeze-drying chamber after cooling to −50 ° C. was heated to −10 ° C., and a container filled with the solution was placed. After standing overnight, the mixture was cooled to −20 ° C. or lower, and further, as a turbidity control, a container filled with the same liquid was received and completely frozen, and then a freeze-dried preparation was prepared by a method of reducing pressure and drying.

Lyophilized preparation pretreated at -6 ° C
Dissolve sodium phosphate so that it contains IL-11 5 mg / ml, glycine 300 mM, and phosphoric acid 10 mM, adjust the pH to 7, fill the container (glass ampule), freeze-dry in advance and freeze-dry The shelf in which ice nuclei were formed in the refrigerator was heated to −6 ° C., and the others were operated in the same manner as in Example 2 to prepare a freeze-dried preparation.

Freeze-dried preparation pretreated at −2 ° C. A freeze-dried preparation was prepared by the same treatment as in Example 3 except that it was frozen at −2 ° C.

Freeze-dried preparation that has been pretreated and frozen under reduced pressure
Dissolve sodium phosphate so that it contains IL-11 5mg / ml, glycine 300mM and phosphoric acid 10mM, adjust the pH to 7 and fill the container (glass ampule). After cooling, the inside of the freeze-drying chamber was decompressed and frozen at a solution temperature of −20 ° C. or higher. Then, after cooling to -20 ° C. or lower, a freeze-dried preparation was prepared by a method of reducing pressure and drying.

(Experimental example 1)
Hereinafter, comparative experimental examples are shown for the freeze-dried preparations prepared in Examples 1 to 5 and Reference Examples 1 to 3 of the present invention.
[Test method 1] Evaluation of turbidity of redissolved solution by absorbance measurement
Water for injection (1.2 mL) is injected into a freeze-dried preparation containing 5 mg of IL-11, and the absorbance at OD650 nm of the redissolved solution after 3, 5, and 7 minutes has been measured. In addition, regarding the turbidity evaluation method of the solution by the absorbance measurement, the following two reports were referred.
(1) Pharmaceutical Research 26 (4) 223-230 (1955) “Examination of Turbidity Evaluation Method in Pharmaceutical Solution Testing”
(2) J. Pharm. Sci. Tech., 48 (2) 64-70 (1994) “A turbidimetric method to determine visual appearance of protein solutions”
(experimental method)
The turbidity control preparation prepared in the same manner as Sample 1 of Reference Example 1 and the preparations prepared in Examples 1 to 5 were redissolved with JP injection water, and the absorbance at a wavelength of 650 nm after 3 minutes was measured with a UV meter. The results are shown in FIG. The turbidity was measured in accordance with Test Method 1. “The degree of turbidity is small” in FIG. 1 described later is a turbidity control preparation prepared in the same manner as Sample 1 with the absorbance after 3 minutes from the addition of the redissolved solution. It means that the absorbance is 0.6 or less.
Further, the properties of the preparations prepared in Reference Samples 1 to 3 and Examples 1 to 5 were observed with the naked eye after 3 and 20 minutes after re-dissolution, and the results are shown in Table 1.
Further, for Sample 1 and Example 3, the appearance of the container was observed after lyophilization and the presence or absence of damage to the container was confirmed. The results are shown in Table 2.

(Experimental result)
FIG. 1 is a graph showing the ratio of the turbidity of 3 minutes after re-dissolution of Examples 1 to 4 to the turbidity of 3 minutes after re-dissolution of the turbidity control preparation prepared in the same manner as Sample 1. Table 1 shows the results of visual observation of Samples 1 to 3 and Examples 1 to 5 at 3 minutes and 20 minutes after redissolving. In all cases, the examples showed better results with respect to turbidity than the samples 1 to 3. Table 2 shows the number of lyophilized samples prepared in Sample 1 and Example 3 and the number of broken containers (glass ampoules). In sample 1, container breakage occurred, while in Example 3, no container breakage was observed.

  As a result, since the redissolved solutions of Samples 1 and 2 became cloudy, this cloudiness was generated by freezing at -20 ° C. or lower and drying regardless of the cooling rate. Moreover, since the sample 3 also became cloudy, even if it added the method of heating up after making it freeze in a freezing process (even if it annealed), the solution state of a formulation was not improved (Table 1). On the other hand, as is clear from the examples, the resolubility of the IL-11 preparations of Examples 1 to 4 is more turbid than the resolubility of the control preparation dried at −20 ° C. or lower after freezing. It was confirmed that the temperature during freezing is desirably set to −20 ° C. to 0 ° C., preferably −15 ° C. to −2 ° C. (FIG. 1). Furthermore, it was confirmed that the re-solubility of the preparation prepared by freezing the solution by reducing the pressure was also good (Table 1). In addition, the preparation produced in Example 3 showed no container breakage, indicating that the production method according to the present invention was a method without fear of container breakage (Table 2).

(Experimental example 2)
Crystallization of sodium phosphate in the lyophilization treatment (Reference Example 1) without pretreatment was detected by DSC (Differential Scanning Calorimetry) (FIG. 2). The horizontal axis represents temperature, and the vertical axis represents the amount of heat generated. An exothermic peak was observed around -18 ° C, and crystallization was observed. In the freezing process, sodium phosphate cannot be crystallized in the presence of an active ingredient such as protein, strongly suggesting that amorphous phosphate ions interact with the active ingredient. Sodium phosphate contains about half of the divalent anion of phosphate-disodium hydrogen phosphate (Na ₂ HPO ₄ , HPO ₄ ²⁻ ) (pH 7.0), and is presumed to be electrostatically bound to the polyvalent cationic main agent. It was done. It was inferred that the binding of the amorphous disodium hydrogen phosphate supplying the divalent anion to the protein of the main drug during freezing leads to deterioration of the solution during re-dissolution and clouding of the solution. As DSC observed crystallization of sodium phosphate with temperature rise during primary drying, it could not be crystallized during the freezing process, and if it could not be crystallized during the freezing process, it was crystallized during the drying process. Was shown to happen.

(Experimental example 3)
To the preparation solution shown in Example 3, a saturated methyl red / ethanol solution was added to a concentration of 0.2%. Furthermore, when freeze-dried preparations were prepared by the methods shown in Example 3 and Reference Example 1, respectively, the freeze-dried preparations prepared by the method of Example 3 turned red when cooled (-27 ° C.). On the other hand, in the sample prepared by the method of Reference Example 1, reddening of the prepared solution was not observed. The red color of the preparation liquid indicates that the preparation liquid was acidified, and thus it was shown that the sodium phosphate in the preparation liquid was crystallized by the pretreatment.

It is the result which showed the ratio of the absorbance for 3 minutes after re-dissolution of Examples 1 to 4 to the turbidity for 3 minutes after re-dissolution of the turbidity control preparation produced in the same manner as Sample 1.

It is a DSC measurement result of sodium phosphate.

Claims (12)

A method for preventing turbidity at the time of re-dissolution of a freeze-dried preparation, comprising means for crystallizing a buffer in the production process of a freeze-dried preparation containing a bioactive protein containing a bioactive protein and a buffer. The method for preventing turbidity at the time of redissolution according to claim 1, wherein the means for crystallizing the buffering agent performs pretreatment under a temperature condition of -20 ° C to 0 ° C before the freeze-drying treatment. The method for preventing turbidity during re-dissolution of a lyophilized preparation according to claim 1 or 2, wherein the physiologically active protein is basic. The method for preventing turbidity during re-dissolution of a lyophilized preparation according to claim 3, wherein the physiologically active protein is interleukin 11. The method for preventing cloudiness at the time of re-dissolution of a lyophilized preparation according to any one of claims 1 to 4, wherein the buffer is sodium phosphate and / or histidine. An ampoule breakage avoiding method for a freeze-dried preparation comprising a means for crystallizing a buffer in a production process of a freeze-dried preparation containing a bioactive protein containing a bioactive protein and a buffer. The ampoule breakage avoiding method according to claim 6, wherein the means for crystallizing the buffering agent performs pretreatment under a temperature condition of -20 ° C to 0 ° C before the freeze-drying treatment. The method for avoiding breakage of an ampoule in a freeze-dried preparation according to claim 6 or 7, wherein the physiologically active protein is basic. The method for avoiding ampule breakage in a freeze-dried preparation according to claim 8, wherein the physiologically active protein is interleukin 11. 10. The ampoule breakage avoidance method for freeze-dried preparations according to claim 6, wherein the buffer is sodium phosphate and / or histidine. A method for producing a lyophilized preparation containing a physiologically active protein comprising the method according to any one of claims 1 to 10. A lyophilized preparation containing a physiologically active protein prepared by the production method according to claim 11.

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