JP2010120966A - Stabilized protein composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production of a composition including a stable IL-2 without including components derived from warm blooded organisms such as human, swine or bovine serum albumin, or carnitine. <P>SOLUTION: The composition including at least IL-2, a sugar and/or a sugar alcohol, an amino acid, and an acid and a base if necessary, and additionally a surfactant if necessary shows scarcely any decline of contents of pertinent drugs even on temporal storage. In addition, the storage of the composition in a silicone-coated container can further inhibit the decline of the content of drugs. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention does not contain a stable protein composition, specifically serum albumin and carnitine derived from warm-blooded animals, and at least interleukin-2, sugar and / or sugar alcohol (except sorbitol), amino acids (glycine and Protein composition (excluding histidine) (excluding compositions containing alanine and sucrose, compositions containing lysine hydrochloride and sucrose), acid and base if necessary, and surfactant if necessary It is about protein composition.

  Interleukin-2 (hereinafter abbreviated as IL-2) is a protein having physiological activity to proliferate T cells and natural killer cells, and is used as a therapeutic agent for various cancers, particularly hemangiosarcoma and renal cancer, and immunodeficiency. There are great expectations. There is also great expectation as a therapeutic agent for acquired immune deficiency syndrome (AIDS). In addition, as with IL-2, as a protein having antitumor activity, there is interferon (hereinafter abbreviated as IFN), which also has antiviral activity induced from animal cells by viruses, double-stranded RNA, lectins and the like. Furthermore, epidermal growth factor (hereinafter abbreviated as EGF) has an action of promoting the differentiation and proliferation of epithelial cells and enhances phosphorylation of intracellular proteins.

Since protein components such as IL-2, IFN and EGF are substances that are unstable under normal storage conditions, various stabilization methods have been attempted. For example, a composition containing IL-2 is obtained by adding a base and an acid to a solution containing IL-2 containing human serum albumin as a stabilizer to neutralize the solution (eg, Patent Document 1) In some cases, an IL-2 composition is obtained by blending with a reducing substance or human serum albumin (eg, Patent Document 2). Further, an IL-2 composition obtained by containing glycine as an amino acid and sucrose as a sugar and adjusted to pH 5 to 6.5 with either histidine (base) or citric acid (acid) (example: Patent Document 3), an IL-2 composition containing a mixture of arginine and carnitine (4-trimethylamino-3-hydroxybutyric acid, also known as vitamin B _T ), sucrose and citric acid (eg, Patent Document 4) ) Among the above-mentioned preparations containing proteins other than IL-2, IFN, and EGF, there are those obtained by stabilizing a protein with a sugar and an amino acid (eg, Patent Documents 5, 6 and 7).

  However, it has recently been pointed out that serum albumin derived from a warm animal such as human serum albumin (HSA) has a risk of viral infection, mad cow disease and the like. It is hoped that it will not. In addition, IL-2 is relatively hardly soluble in water, and since an agglutination reaction usually occurs in the vicinity of neutrality and the solubility is lowered. After dissolving the IL-2 by adding a base or acid, the acid or base It is necessary to add near neutral. For this reason, as described in Patent Document 3, if only one of an acid and a base is added, an agglutination reaction occurs, and the solubility of IL-2 may be reduced. Furthermore, carnitine as a stabilizer disclosed in Patent Document 4 is a thyroid inhibitor and may cause side effects depending on the amount added. In addition, as for protein stabilization methods, it is described in the literature that, in general, if the type of protein is different, it cannot always be stabilized similarly [Non-Patent Documents 1 and 2], and the above-mentioned IL- 2 or a composition capable of stabilizing a protein component different from IFN does not always stabilize the protein component.

JP-A-62-164631 JP-A-60-215631 WO02 / 00243 WO90 / 00397 Special table flat 8-504784 WO96 / 41642 Special table 2001-503781 WO02 / 00243

Int. J. et al. Pharm. 185, 129-188 (1999) Int. J. et al. Pharm. 203, 1-60 (2000)

  Thus, a composition having high safety and good storage stability has been desired as a protein component.

  In view of the above circumstances, the present inventors do not include serum albumin and carnitine derived from warm-blooded animals, and are pharmaceutically acceptable sugars and / or sugar alcohols, amino acids, and if necessary, acids and bases, and if necessary. The composition which improved stability of IL-2 by mix | blending surfactant etc. was discovered, and this invention shown below was completed.

(1) Warm-blooded animal-derived serum albumin and carnitine are not blended, and at least the following components 1) interleukin 2 in a ratio of 0.1 to 100 μg per ml of protein composition solution,
2. A protein composition characterized by containing sucrose and 3) arginine (except for a composition containing alanine and sucrose, and a composition containing lysine hydrochloride and sucrose).

(2) Serum albumin and carnitine derived from warm-blooded animals are not blended, and at least the following components 1) Interleukin 2 in a ratio of 40 μg per ml of protein composition solution,
2. A protein composition characterized by containing sucrose and 3) arginine (except for a composition containing alanine and sucrose, and a composition containing lysine hydrochloride and sucrose).

  (3) The sucrose is contained at a rate of 5 to 500 mg per 1 mL of the protein composition solution, or arginine is contained at a rate of 0.1 to 250 mg per 1 mL of the protein composition solution. Or a protein composition according to (2).

  (4) The protein composition according to any one of (1) to (3), further comprising an acid or a base.

  (5) The protein composition according to any one of (1) to (4), further comprising a surfactant.

  (6) The protein composition according to any one of (1) to (5), which is a freeze-dried product.

(7) Containing no serum albumin and carnitine derived from warm-blooded animals, and at least the following components 1) interleukin 2,
The method for producing a protein composition according to any one of (1) to (6), comprising a step of freeze-drying a solution containing 2) sucrose and 3) arginine.

  (8) A step of adding a base to the solution to adjust the pH to 8 to 11, and then adding an acid to neutralize, or adding an acid to the solution to adjust to a pH of 2 to 6, and then adding a base to neutralize the solution. The manufacturing method as described in (7) characterized by including.

  (9) The production method according to (7) or (8), wherein the solution further contains a surfactant.

  The composition containing IL-2 of the present invention can be used as a solution (eg, injection) in which IL-2 and the following additives are dissolved. The liquid medium of the solution is dried to obtain a solid agent (eg, : Freeze-dried preparation). If it is a solid preparation, the composition can be stored for a long period of time. In the present invention, all naturally-derived or recombinant IL-2 can be used as IL-2. In particular, recombinant human IL-2 is preferable.

  Even when the concentration of IL-2 in the composition is low, it is a great feature that the drug is hardly adsorbed on the container wall surface. When the composition of the present invention is used as a solution, IL-2 is contained in 1 mL of the solution. May be blended in an amount of 0.1 to 100 μg, preferably 0.5 to 100 μg, more preferably 1 to 100 μg. Moreover, when using the composition of this invention as a solid agent, IL-2 is 0.001-5 w / w% with respect to solid agent whole quantity, Preferably it is 0.0025-2.5 w / w%, More preferably What is necessary is just to mix | blend 0.003-1 w / w%. If the IL-2 content is less than the above blending amount, the medicinal effect may not be sufficiently exhibited. If the amount is more than the above-mentioned blending amount, there is a possibility that a drug such as IL-2 is not completely dissolved in the solution.

  In the present invention, sugar and / or sugar alcohol means monosaccharide, disaccharide, polysaccharide or water-soluble glucan. Sugar and / or sugar alcohol is added for the stabilization of IL-2, but can also be added as a solubilizer, excipient or isotonic agent. Specific examples of the sugar include glucose, mannose, sorbose, xylose, maltose, lactose, fructose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, carboxymethylcellulose-Na and the like. The sugar alcohol is preferably a C4 to C8 sugar alcohol, and specific examples include mannitol, inositol, dulcitol, xylitol, arabitol, raffinose, erythritol, maltitol, lactitol, palatinit, trehalose and the like. Among the sugars and / or sugar alcohols, maltose, mannitol, lactose, sucrose and sorbitol are preferable, maltose, mannitol, lactose and sucrose are more preferable, and sucrose is particularly preferable.

  The sugars and / or sugar alcohols can be used alone or in a mixture. The blending amount is not particularly limited as long as it is an amount that can be dissolved in the solution of the composition and enhance the stability of IL-2. The mixing ratio of sugar and / or sugar alcohol to IL-2 may be 1 to 20000 times, preferably 5 to 17500 times, more preferably 20 to 15000 times by weight. When the composition of the present invention is used as a solution, 5 to 500 mg, preferably 10 to 250 mg, more preferably 20 to 200 mg of sugar and / or sugar alcohol may be added to 1 mL of the solution of the composition. If the amount is less than the above amount, IL-2 may not be stabilized. If the amount is too large, the IL-2 content relative to the total amount of the composition is relatively decreased, and a large amount of IL-2 composition is administered. Otherwise, there may be no medicinal effects.

  In the present invention, amino acids are added for the stabilization of IL-2, but can also be added as an excipient. An amino acid means a compound having an amino group and a carboxyl group in the molecule, but also includes imino acids such as proline and hydroxyproline. Examples of amino acids include neutral amino acids, acidic amino acids, and basic amino acids. Specifically, as neutral amino acids, alanine, valine, norvaline, leucine, norleucine, isoleucine, phenylalanine, tyrosine, diiodotyrosine, sulamine, threonine, serine, proline, hydroxyproline, tryptophan, thyroxine, methionine, cystine, cysteine, Examples include α-aminobutyric acid. Examples of acidic amino acids include aspartic acid, glutamic acid, asparagine, and glutamine. Examples of basic amino acids include lysine, lysine hydrochloride, arginine, hydroxylysine and the like. Among the amino acids, alanine, lysine hydrochloride and arginine are preferable, and arginine is more preferable.

  The aforementioned amino acids can be used alone or in a mixture. The addition amount is not particularly limited, and may be any amount that can be dissolved in the solution of the composition and increase the stability of IL-2. The mixing ratio of the amino acid with respect to IL-2 may be 1 to 10,000 times, preferably 10 to 7500 times, more preferably 20 to 5000 times by weight. When the composition of the present invention is used as a solution, 0.1 to 250 mg, preferably 0.5 to 125 mg, more preferably 1 to 100 mg of amino acid may be added to 1 mL of the solution of the composition. If the added amount is less than the above amount, IL-2 may not be stabilized. If the added amount is large, the content of IL-2 relative to the total amount of the composition is relatively lowered, and a composition containing a large amount of IL-2 is administered. Otherwise, there may be no medicinal effects.

  As described above, the composition of the present invention contains a sugar and / or a sugar alcohol and an amino acid, and therefore may cause a Maillard reaction in a solid state or a liquid state. The Maillard reaction is a reaction in which amino groups of amino acids, peptides and proteins react with ketones, aldehydes, particularly reducing sugars, to produce brown pigments. However, in the composition of the present invention, in particular, when the sugar and / or sugar alcohol is sucrose and the amino acid is in combination with arginine, the Maillard can be stored even if the composition is stored at 40 ° C. for about 30 days. The reaction is less likely to occur and the possibility of reducing the content of IL-2 is low. However, the composition containing alanine and sucrose and the composition containing lysine hydrochloride and sucrose melted the freeze-dried product, and the dried product could not be produced.

  The ratio of the content of the sugar and / or sugar alcohol to the amino acid is 0.01 to 4 times, preferably 0.025 to 3 times, more preferably 0, in terms of the weight ratio of the amino acid to the sugar and / or sugar alcohol. 0.05 to 2 times. In the case of protein preparations, the presence of the Maillard reaction is different if the main protein is the same even if the additive is the same. Therefore, in order to prevent the Maillard reaction from occurring, the additive in the preparation depends on the type of protein. It is necessary to consider the type and quantity.

  In the present invention, an acid may be blended if necessary. As the acid, any physiologically acceptable acid can be used. Examples include organic acids such as acetic acid, lactic acid, succinic acid, tartaric acid, citric acid, aspartic acid, glutamic acid, asparagine, and glutamine, and inorganic acids such as hydrochloric acid and phosphoric acid. Of these, citric acid and tartaric acid are preferred. These pharmacologically acceptable salts may also be used. Although the addition amount of an acid changes with kinds of acid to be used, what is necessary is just to add the necessary amount which can be adjusted to desired pH. Generally, the blending ratio of the acid to IL-2 is 5 to 12000 times, preferably 10 to 6000 times, more preferably 25 to 3000 times by weight. When the composition of the present invention is used as a solution, 0.25 to 50 mg, preferably 0.5 to 20 mg, more preferably 1 to 10 mg of acid may be added to 1 mL of the solution of the composition. Moreover, if it is less than the said amount, pH will become basic, and if it is more, pH will become acidic, and there is a possibility that IL-2 cannot be stabilized in any state.

  Among organic acids, acidic amino acids aspartic acid, glutamic acid, asparagine, and glutamine can be amino acids as well as acids in the constituent components.

  In the present invention, a base may be blended if necessary. As the base, any physiologically acceptable base can be used. For example, alcohol amines such as N-methylglucamine, monoethanolamine, diethanolamine and triethanolamine, alkylamines such as mono, di or triethylamine, basic amino acids such as arginine, lysine and lysine hydrochloride, and inorganics such as sodium carbonate A base or the like is used, and these may be used alone or in a mixture of two or more. When an inorganic base such as sodium carbonate is used, it is preferably used in combination with the above amine. Preferred bases are N-methylglucamine, diethanolamine, triethanolamine, and arginine, and more preferred are diethanolamine and arginine. The bases may be used alone or in a mixture of two or more. Although the addition amount of a base changes also with the kind of base to be used, what is necessary is just to add the required quantity which can be adjusted to desired pH. Generally, the blending ratio of the base to IL-2 is 2 to 10,000 times, preferably 5 to 5000 times, more preferably 10 to 4500 times by weight. When the composition of the present invention is used as a solution, 0.1 to 100 mg, preferably 0.25 to 100 mg, more preferably 1 to 50 mg of acid may be added to 1 mL of the solution of the composition. Further, when the composition of the present invention is used as a solid agent, if the amount is less than the above amount, the pH of the solution becomes acidic, and if it is more, the pH of the solution becomes basic, and IL-2 is stabilized in any state. There is a fear that it cannot be done.

  The basic amino acids arginine, lysine, lysine hydrochloride, and the like can be amino acids as well as constituent bases.

  In the present invention, a surfactant may be added if necessary. As the surfactant, any physiologically acceptable surfactant may be used, and by adding the surfactant, the solid agent is redissolved in distilled water at the time of preparing the composition solution before drying in the preparation of the solid agent. Improved solubility of IL-2 and improved stability of IL-2 when the liquid medium of the composition solution is dried (especially the composition prevents the adsorption of the wall surface of the IL-2 container during drying) The effect of suppressing the decrease of the IL-2 content in the medium is obtained. Any surfactant can be used as long as it can be dissolved or suspended in a solution of the composition and can enhance the stability of IL-2, but is preferably a nonionic surfactant, specifically a polyoxyethylene cured agent. Castor oil, sucrose fatty acid ester, polyethylene glycol (PEG), polyoxyethylene polyoxypropylene glycol, polyoxyethylene sorbitan fatty acid ester and the like. Specifically, polyoxyethylene hydrogenated castor oil 5, polyoxyethylene hydrogenated castor oil 10, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 20, polyoxyethylene hydrogenated castor oil 40, polyoxyethylene hydrogenated castor oil 50, polyoxyethylene hydrogenated castor oil 60 and the like. Specific examples of polyethylene glycol include Macrogol 200, Macrogol 300, Macrogol 400, Macrogol 600, Macrogol 1000, Macrogol 1500, Macrogol 1540, Macrogol 2000, Macrogol 4000, Macrogol 6000, Macrogol There are 20000 etc. Specifically, as polyoxyethylene polyoxypropylene glycol, polyoxyethylene (105) polyoxypropylene (5) glycol, polyoxyethylene (120) polyoxypropylene (40) glycol [Pluronic F87], polyoxyethylene (160 ) Polyoxypropylene (30) glycol [Pluronic F68], Polyoxyethylene (42) Polyoxypropylene (67) Glycol [Pluronic P123], Polyoxyethylene (54) Polyoxypropylene (39) Glycol [Pluronic F85], Poly Oxyethylene (196) polyoxypropylene (67) glycol [Pluronic F127], polyoxyethylene (20) polyoxypropylene (20) glycol [Pluronic L44 And the like. Specific examples of the polyoxyethylene sorbitan fatty acid ester include polysorbate 20 [Tween 20], polysorbate 40 [Tween 40], polysorbate 60 [Tween 60], polysorbate 65 [Tween 65], polysorbate 80 [Tween 80], and the like. More preferred is polyoxyethylene sorbitan fatty acid ester, and particularly preferred is polysorbate 80 [Tween 80].

  The blending amount of the surfactant varies depending on the type of the surfactant used, but the blending ratio of the surfactant to IL-2 is 1 to 300 times, preferably 2.5 to 250 times, more preferably 5 by weight. What is necessary is just to be -200 times. When the composition of the present invention is used as a solution, 0.05 mg to 15 mg, preferably 0.075 mg to 7.5 mg, more preferably 0.1 mg to 4 mg may be added to 1 mL of the composition solution. In addition, when the composition of the present invention is used as a solid agent, the solubility of IL-2 may not be improved and the stability of IL-2 may be lowered if the amount is less than the above-mentioned addition amount. For example, during preparation, it may be difficult to prepare the composition solution due to foaming.

  In the present invention, it is preferable to use a buffering agent in order to minimize pH fluctuations during the preparation of the composition solution before drying in the preparation of the solid agent or when the solid agent is redissolved in distilled water. The buffering agent may be any physiologically acceptable buffering agent, and examples thereof include phosphate-based and citrate-based buffering agents.

  The addition amount of the buffering agent varies depending on the type of the buffering agent to be used, but an amount necessary for maintaining the desired pH, that is, the pH of the solution of the composition at 6.1 to 9 may be used. Specifically, the blending ratio of the buffer to IL-2 may be 20 to 10,000 times, preferably 50 to 8000 times, more preferably 100 to 6000 times by weight. When the composition of the present invention is used as a solution, 1 to 100 mg, preferably 2.5 to 75 mg, more preferably 5 to 50 mg of a buffer may be added to 1 mL of the solution of the composition. If the amount is less than the above amount, there may be a large pH fluctuation during the production of the composition solution or when the solid agent is redissolved in distilled water. If the amount is large, the content of IL-2 relative to the total amount of the composition is relatively lowered. However, if a large amount of IL-2 composition is not administered, there is a possibility that the medicinal effect does not occur.

  The preparation of the present invention may contain pharmaceutically acceptable additives depending on the form of the preparation. For example, in the case of injections, preservatives such as phenol and cresol, sulfites, sodium pyrosulfite, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), antioxidants such as thioglycolic acid, soothing agents such as benzyl alcohol and chlorobutanol Agents, isotonic agents such as sodium chloride and glycerin may be added.

  When a serum albumin derived from a warm-blooded animal such as human serum albumin, bovine serum albumin, porcine serum albumin, etc. is added as a stabilizer, side effects may occur as described above. Recombinant albumin has a low possibility of causing the above side effects and may be added as an additive of the composition. The addition of recombinant albumin is useful for preventing a decrease in IL-2 stability. The blending ratio of the recombinant albumin to IL-2 may be 1 to 4000 times, preferably 2.5 to 2000 times, more preferably 5 to 1000 times by weight. When the composition of the present invention is used as a solution, 0.1 mg to 100 mg, preferably 0.25 mg to 50 mg, more preferably 0.5 mg to 25 mg may be added to 1 mL of the composition solution.

  The composition of the present invention comprises at least one of IL-2, sugar and / or sugar alcohol, amino acid, acid and base if necessary, surfactant if necessary, and buffer for adjusting pH. Contains compounding ingredients such as agents. As a preferable combination of the components of these compositions, a combination of IL-2, sucrose, arginine, citric acid, diethanolamine (may not include diethanolamine in some cases), and if necessary, a combination of polysorbate 80 and phosphate buffer It is a composition. About the compounding quantity of the compounding component of this preferable combination, IL-2 is 0.001-5 w / w% with respect to solid agent whole quantity, Preferably it is 0.005-2.5 w / w%, More preferably, 0.01- 1 w / w%, the blending ratio of sucrose to IL-2 is 1 to 10,000 times, preferably 5 to 5000 times, more preferably 20 to 4000 times by weight, and the blending ratio of arginine to IL-2 is 1 by weight. To 5000 times, preferably 10 to 2500 times, more preferably 20 to 2000 times, and the mixing ratio of citric acid to IL-2 is 5 to 2000 times, preferably 10 to 1000 times, more preferably 25 to 500 times by weight. The mixing ratio of diethanolamine to IL-2 is 10 to 5000 times, preferably 25 to 2500 times, more preferably 50 to 2000 times (by weight). In some cases, diethanolamine may not be blended), and the blending ratio of polysorbate 80 to IL-2 is 1 to 300 times, preferably 2.5 to 250 times, more preferably 5 to 200 times, If an acid buffer is added, it may be added at a blending ratio necessary to maintain the pH at 6.1 to 9, but specifically, the blending ratio of the buffer to IL-2 is 20 to 20 by weight. It may be 2000 times, preferably 50 to 1500 times, more preferably 100 to 1000 times. When the composition of the present invention is used as a solution, IL-2 is 0.1 to 100 μg, preferably 0.5 to 100 μg, more preferably 1 to 100 μg, and sucrose in 1 mL of the composition solution. 5 to 500 mg, preferably 10 to 250 mg, more preferably 20 to 200 mg in 1 mL of the solution of arginine, 0.1 to 250 mg, preferably 0.5 to 125 mg, more preferably 1 in 1 mL of the solution of the composition. -100 mg, citric acid is 0.25-50 mg, preferably 0.5-20 mg, more preferably 1-10 mg, in 1 mL of the composition solution, and diethanolamine is 0.1-0.1 mg in 1 mL of the composition solution. 100 mg, preferably 0.25 to 100 mg, more preferably 1 to 50 mg (in some cases, diethanolamine may not be added) If polysorbate 80 is added, 0.05 mg to 15 mg, preferably 0.075 mg to 7.5 mg, more preferably 0.1 mg to 4 mg, phosphate buffer is added to 1 mL of the composition solution. Then, it may be added at a blending ratio necessary to maintain the pH 6.1-9, specifically, 1-100 mg, preferably 2.5-75 mg, more preferably in 1 mL of the composition solution. What is necessary is just to add 5-50 mg. When the composition of the present invention is used as a solid agent, the ratio of the content of sucrose and arginine is 0.01 to 4 times, preferably 0.05 to 3 times, by weight of arginine with respect to sucrose. More preferably, it is 0.1 to 2 times.

  As a method for drying the solution in producing the solid agent from the solution of the present invention, it is only necessary to dry the liquid medium of the composition solution, but preferably a freeze drying method, a fluidized bed drying method, a spray drying method, and more preferably. There is a freeze-drying method. In the case of a preparation containing a protein component such as IL-2, a freeze-dried product that has been solidified by a freeze-drying method is not subjected to stress such as heat and can produce a stable protein preparation.

  If the pH of the composition solution is adjusted to about 6 or more, IL-2 can be kept stable, and the liquid state during the drying operation of the liquid medium and when the solid preparation is redissolved can be kept transparent. Thus, for the purposes of the present invention, there is no particular upper limit to the pH, but preferably the final pH adjustment is pH 6.1-9 in solution. When the composition of the present invention is used as an injection, the composition is preferably adjusted in the vicinity of a physiological pH range. In this case, the final pH range is pH 6.1 to 8, more preferably pH 6 5 to 7.5. On the acidic side of the lower limit pH, it may be difficult to keep the liquid state at the time of re-dissolution transparent, and on the basic side of the upper limit pH, the stability of the composition and the transparency of the solution are satisfactory. However, there is a possibility that irritation to the skin, blood vessels and the like may increase, which is not always preferable.

  The reason why the stability of IL-2 has increased in the present invention is not necessarily clear, but 1) decomposition of IL-2, 2) aggregation of IL-2, 3) adsorption of IL-2 to a container, etc. This is thought to be due to suppression. In particular, when the drug concentration in the preparation is low, the effect of drug adsorption on the container wall is large, but with the present invention formulation, it is possible to suppress adsorption of IL-2 to the container at the time of drying. . In addition, the IL-2 content immediately after the production of the composition hardly decreases even after storage for 30 days, for example, at 40 ° C. for about 30 days. In order to further suppress the adsorption of IL-2 to the container, various methods are conceivable. One method is to contain a composition containing IL-2 in a container whose inner wall is coated with silicone. .

  Although the manufacturing method of this invention IL-2 composition is not specifically limited, Preferably it manufactures with the following methods. An appropriate amount of the IL-2 stock solution and, if necessary, a surfactant is added to a solution in which the necessary amount of sugar and / or sugar alcohol and amino acid as a stabilizer is dissolved in distilled water. Next, an appropriate amount of a base (when a basic amino acid is added as an amino acid, a base may not be added) is added to a pH of about 8 to 11, preferably about pH 9 to 11, more preferably about pH 9. 5 to 10.5, and after adding a buffer solution, quickly add an acid to a pH of about 7 to 7.5. The remaining distilled water is added to adjust the amount of the solution, and after the composition solution is produced, it is sterile filtered, dispensed into a container, and then freeze-dried. Alternatively, an appropriate amount of IL-2 and, if necessary, a surfactant is added to a solution in which the necessary amount of sugar and / or sugar alcohol and amino acid as a stabilizer is dissolved in distilled water. Next, an appropriate amount of acid (when an acidic amino acid is added as an amino acid, an acid may not be added) is added, and once the pH is about 2 to 6, preferably about pH 2 to 4, more preferably about pH 2 After adding a buffer solution, a base is quickly added to obtain a pH of about 7 to 7.5, and a composition is produced in the same manner as described above. Among the production methods, the former production method is preferable from the viewpoint of the stability of IL-2. In lyophilization, the solution of the composition prepared above is rapidly frozen at about −60 ° C. to about −10 ° C., preferably about −50 ° C. to about −40 ° C., and if necessary, while supplying sublimation heat, Preferably, moisture is sublimated and removed for 48 to 72 hours until 0.005 to 1 mb is reached, and if necessary, inert gas such as nitrogen or dry air is filled and sealed.

  In the above production method, a base or an acid is added to obtain a required pH, and then the "rapidly" acid or base is added to adjust the pH to about 7 to 7.5. The time from the start of addition until the pH is adjusted to about 7 to 7.5 varies depending on the production amount of the composition, but may be within about 60 minutes, preferably about 45 minutes, more preferably within about 30 minutes. .

  The composition of the present invention does not contain serum albumin and carnitine derived from warm-blooded animals, and contains at least IL-2, sugar and / or sugar alcohol, amino acid, and if necessary, acid and base components. For example, IL-2 can be stably stored for a long period of time, for example, at about 40 ° C. for 30 days by blending a surfactant, a buffering agent, or the like. Furthermore, the preparation of the present invention can suppress the adsorption of the IL-2 container wall surface.

  Although the usage method of this invention composition is not specifically limited, It is preferable to use parenterally. When used as an injection, the freeze-dried composition is dissolved at the time of use in distilled water for injection, physiological saline, glucose solution, appropriate infusion solution, and the like, and is intravenously, intramuscularly, subcutaneously or intradermally. To be administered. In addition, a suitable carrier, excipient or the like may be added to the present composition to prepare a preparation for topical administration such as oral, nasal or intrathecal administration. As a dosage, for example, IL-2 is 350,000 to 1.4 million JRU (national standard unit) per day.

  The present invention will be described more specifically with reference to the following examples. However, this is merely an example and does not limit the present invention.

  The titer of IL-2 used in the present invention is a method of calculating the unit of IL-2 using the proliferation of NK cells (derived from mouse cells) that proliferates in an IL-2-dependent manner as an index for quantification. And measured. NK-7 cells, a type of NK cell, proliferate in proportion to the amount of substance having IL-2 activity. When MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] is given to NK-7 cells, MTT is taken up by NK-7 cells, Reduced by two electron carriers associated with the respiratory chain present in the membrane, cytochrome b and cytochrome c, yields an amount of dye formazan proportional to the proliferative capacity of the cell. The titer of IL-2 is determined from the relationship between the amount of formazan produced and the dilution factor of the sample. The produced formazan was eluted from NK-7 cells with isopropanol, and the titer of IL-2 was measured by measuring the absorbance of purple-colored liquid (wavelength 560 nm).

(Example 1) Examination of amino acids and sugars capable of lyophilization (not including IL-2)
According to the manufacturing method shown below, the composition of the component shown in Table 1 was manufactured, and the external appearance of the composition was observed. In addition, IL-2 which is a main ingredient is not mix | blended with this composition.

(Example 1 preparation method)
As sugar or sugar alcohol, maltose, mannitol, lactose or sucrose was used. Alanine, lysine hydrochloride, and arginine were used as amino acids. Polysorbate 80 was used as the surfactant, diethanolamine as the base, citric acid as the acid, and phosphate buffer as the buffer.

  120 mL / mL sugar or sugar alcohol aqueous solution 5 mL, 10 mg / mL polysorbate 80 aqueous solution 0.5 mL, and 100 mg / mL amino acid aqueous solution 2 mL were mixed and dissolved. About 0.3 mL of a 100 mg / ml diethanolamine aqueous solution was added to the solution to adjust the pH to about 9. In addition, diethanolamine is not added in the arginine addition formulation. Next, 14.6 mg of phosphate buffer and about 1.48 mg of citric acid were added to adjust the pH of the solution to about 7, and the total volume was adjusted to 10 mL with water for injection. Thereafter, 1 mL of the prepared composition solution and 2 mL of a solution prepared by mixing 1 mL of the prepared composition solution and 1 mL of distilled water for injection were each dispensed into 3 mL glass vials and freeze-dried. Freeze-drying was performed under the conditions shown in Table 2.

(Test method)
The appearance of the freeze-dried product immediately after freeze-drying and the freeze-dried product after storage at 40 ° C. for 10 days were visually observed. In Tables 3 and 4 below, the fact that there is no problem in appearance means that the product is a white, homogeneous lyophilized product and does not allow thawing, and the cake is a matrix of the lyophilized product. If this cake is formed, it is stable as a freeze-dried product.

(Test results)
Appearances of the freeze-dried product immediately after freeze-drying and the freeze-dried product after storage at 40 ° C. for 10 days are shown in Tables 3 and 4. As a result, after storage at 40 ° C. for 10 days, when the sugar or sugar alcohol is maltose, the amino acid is a combination of alanine, lysine hydrochloride and arginine. When the sugar or sugar alcohol is mannitol, the amino acid is a combination of alanine, lysine hydrochloride and arginine. When the sugar or sugar alcohol was lactose, the appearance of the composition in which the amino acid was a combination of alanine, lysine hydrochloride and arginine, and when the sugar or sugar alcohol was sucrose, the appearance of the composition in which the amino acid was arginine was particularly good. On the other hand, preparations containing glycine, most compositions containing sorbitol, compositions containing alanine and sucrose, and compositions containing lysine hydrochloride and sucrose are stored at 40 ° C. for 10 days. Did not form.

(Example 2) Examination of amino acids and sugars capable of lyophilization (including IL-2)
In Example 1, each composition was produced in accordance with the production method shown below for a combination of an amino acid and a sugar or sugar alcohol, which had a good appearance of the freeze-dried product. -2 content and appearance of lyophilized product. In addition, IL-2 which is a main ingredient is mix | blended with this formulation.

(Example 2 preparation method)
Table 5 shows the components of the composition. As sugar or sugar alcohol, maltose, mannitol, lactose or sucrose was used. Alanine, lysine hydrochloride, and arginine were used as amino acids. Polysorbate 80 was used as the surfactant, diethanolamine as the base, citric acid as the acid, and phosphate buffer as the buffer.

  0.6 mL of an active ingredient stock solution containing 0.04 mg (700,000 JRU) IL-2, 5 mL of 120 mg / mL sugar or sugar alcohol aqueous solution, 0.5 mL of 10 mg / mL polysorbate 80 aqueous solution, 2 mL of 100 mg / mL amino acid aqueous solution Were mixed and dissolved. About 0.3 mL of a 100 mg / mL diethanolamine aqueous solution (when adding arginine as an amino acid, do not add diethanolamine) is added to the solution to adjust the pH to about 9. Subsequently, 14.6 mg of phosphate buffer and about 1.48 mg of citric acid were added to adjust the pH of the solution to about 7, and the total amount of the solution was adjusted to 10 mL using water for injection. Thereafter, 1 mL of the prepared composition solution and 2 mL of a solution prepared by mixing 1 mL of the manufactured composition solution and 1 mL of distilled water for injection were dispensed into a 3 mL glass vial and lyophilized. Freeze-drying is the same as in Example 1.

(Test method)
The IL-2 content of the freeze-dried product immediately after freeze-drying and the freeze-dried product after storage at 40 ° C. for 14 days was measured, and the appearance of the freeze-dried crystals (only the freeze-dried product after storage at 40 ° C. for 14 days) was visually observed. did. The IL-2 content in the IL-2 lyophilized product was measured by the HPLC method. The freeze-dried product was redissolved with 1 mL of water for injection and used as a measurement sample for the HPLC method. The column was COSMOSIL 5C18-300 (150 × 4.6 mm, average particle diameter of resin in the column was 5 μm, manufactured by Nacalai Tesque), and UV having a wavelength of 220 nm was used for detection of IL-2. The mobile phases are A) 0.1% trifluoroacetic acid-containing water / acetonitrile (95/5), B) 0.07% trifluoroacetic acid-containing acetonitrile. Flowed (gradient method). The osmotic pressure was measured according to the Japanese Pharmacopoeia 14th revised general test method “osmotic pressure measurement method”.

  The content% of IL-2 was calculated by the following formula.

(Test results)
Table 5 Freeze-dried product immediately after freeze-drying and IL-2 content of freeze-dried product after storage at 40 ° C for 14 days, appearance of freeze-dried product (only after freeze-dried product stored at 40 ° C for 14 days) and freeze-dried product Table 7 shows the osmotic pressure and the pH of the solution state when dissolved in water (volume of dissolved distilled water: 1 mL).

  As a result, if the sugar or sugar alcohol is sucrose and the amino acid is a combination of arginine, the IL-2 content is almost the same immediately after lyophilization and after storage for 14 days at 40 ° C., and IL-2 is stabilized. It became clear that. Moreover, in the case of the composition of the above combination, it was presumed that the Maillard reaction did not occur because the freeze-dried product was not colored. Furthermore, in the case of the above combination, the osmotic pressure is about 1, and the pH is adjusted to about 7 and about physiological pH, and even when applied as an injection, there is no risk of pain in the body, etc. Conceivable. In addition, when any lyophilized product of the formulations 1 to 8 was redissolved with distilled water or the like, the liquid state was colorless and clear.

Example 3 Bioassay of IL-2 Composition The IL-2 content in the composition was measured by a bioassay.

(Test method)
Among the lyophilized products shown in Example 2, a lyophilized product in which the decrease in IL-2 content was small after storage over time, that is, a frozen product containing maltose, mannitol, lactose, sucrose as a sugar or sugar alcohol, and arginine as an amino acid. IL-2 in the dried product (formulations 5 to 8 in Example 2) was measured by a bioassay. Manufactured with the formulation components and the production method of Example 2 except that IL-2 was 0.02 mg (350,000 JRU) among the blended components of the lyophilized product. As the freeze-dried product used, a freeze-dried product immediately after freeze-drying and a freeze-dried product stored at 40 ° C. for 30 days were used.

  The bioassay was performed by the following method. This method uses NK-7 cells (derived from a mouse) that proliferates depending on IL-2, and measures the titer from the proliferative ability. NK-7 cells proliferate in proportion to the amount of substance having IL-2 activity. When MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide] is given to NK-7 cells, MTT is taken up by NK-7 cells, Reduced by two electron carriers associated with the respiratory chain present in the membrane, cytochrome b and cytochrome c, yields an amount of dye formazan proportional to the proliferative capacity of the cell. From the relationship between the amount of formazan produced and the dilution factor of the sample, the titer of IL-2 is quantified. The produced formazan was eluted from NK-7 cells with isopropanol and quantified by measuring absorbance (wavelength 560 nm) of a purple colored solution.

(Test results)
The appearance of the lyophilized product after storage at 40 ° C. for 30 days and the results of the bioassay are shown in Table 8 as the ratio of IL-2 activity immediately after lyophilization to IL-2 activity after storage at 40 ° C. for 30 days. As a result, it became clear that the IL-2 activity ratio was almost 1 in any formulation, and the IL-2 activity hardly changed even when stored over time.

(Example 4) Content measurement of IL-2 when the container for the composition was changed The container containing the composition was changed from a glass vial to a vial in which the inner wall surface of the container was coated with silicone. The content was measured.

(Test method)
Among the lyophilized products shown in Example 2, the lyophilized product in which the decrease in IL-2 content was small even after storage over time, that is, sugars or sugar alcohols containing maltose, mannitol, lactose, sucrose, and arginine as amino acids. The IL-2 content in the lyophilized product (formulations 5 to 8 in Example 2) was measured. Manufactured by the blending components and production method of Example 2 except that IL-2 was 0.02 mg (350,000 JRU) among the blended components of the lyophilized product. 1 mL of the solution containing the blended components was dispensed into 3 mL silicone-coated vials and 3 mL glass vials, respectively, and lyophilized. The IL-2 content in the freeze-dried product was measured after freeze-drying once and after freeze-drying twice (re-dissolved in 1 mL of distilled water for injection and freeze-dried after the first freeze-drying). The content measurement method is as shown in Example 2.

(Test results)
The content of IL-2 is shown in Table 9 in JRU units per mL. As a result, in any lyophilized product, the IL-2 content of the lyophilized product contained in the silicone-coated vial was slightly higher than in the glass vial, and the composition was contained in a silicone-coated container. It was revealed that the decrease in IL-2 content was slightly suppressed.

  The present invention relates to an IL-2 containing solution obtained by the preparation as described above, a lyophilized preparation obtained by lyophilizing the obtained solution, and a suitable restoring agent such as distilled water for injection in the lyophilized preparation. Provides various forms of IL-2 compositions, such as solution formulations to the required concentration. Even if these compositions of the present invention do not contain serum albumin derived from warm-blooded animals or carnitine which is a thyroid inhibitor, there is little substantial loss of IL-2 during the production process and storage. Furthermore, the freeze-dried preparation of IL-2 has excellent characteristics such as the liquid state upon re-dissolution is transparent and the adsorption to the container wall is small. Moreover, since the composition of the present invention can be adjusted to near physiological pH, undesirable local effects such as pain at the time of injection and inflammation at the injection site can be avoided.

Claims (9)

Serum albumin and carnitine derived from warm-blooded animals are not blended, and at least the following components 1) interleukin 2 in a ratio of 0.1 to 100 μg per ml of protein composition solution,
2. A protein composition characterized by containing sucrose and 3) arginine (except for a composition containing alanine and sucrose, and a composition containing lysine hydrochloride and sucrose). Serum albumin and carnitine derived from warm-blooded animals are not blended, and at least the following components 1) Interleukin 2 in a ratio of 40 μg per 1 ml of the protein composition solution,
2. A protein composition characterized by containing sucrose and 3) arginine (except for a composition containing alanine and sucrose, and a composition containing lysine hydrochloride and sucrose). The sucrose is contained at a rate of 5 to 500 mg per mL of the protein composition solution, or arginine is contained at a rate of 0.1 to 250 mg per mL of the protein composition solution. The protein composition described in 1. The protein composition according to any one of claims 1 to 3, further comprising an acid or a base. The protein composition according to any one of claims 1 to 4, further comprising a surfactant. The protein composition according to any one of claims 1 to 5, which is a freeze-dried product. Serum albumin and carnitine derived from warm-blooded animals are not blended, and at least the following components 1) interleukin 2,
The method includes a step of lyophilizing a solution containing 2) sucrose and 3) arginine. The manufacturing method of the protein composition in any one of Claims 1 thru | or 6. Adding a base to the solution to adjust the pH to 8 to 11, and then adding an acid to neutralize, or adding an acid to the solution to adjust the pH to 2 to 6, and then adding a base to neutralize the solution. The manufacturing method according to claim 7, wherein the manufacturing method is characterized. The manufacturing method according to claim 7 or 8, wherein the solution further contains a surfactant.