WO1999018119A1 - Process for freeze-drying proteins - Google Patents

Abstract

A process for preparing protein-containing freeze-dried products, characterized by freeze-drying a solution containing a minimum unit of a stable, biologically active protein and a metal salt of an organic acid, while inhibiting the protein from undergoing association to form a polymer in the course of freeze-drying.

Description

 Description Method for freeze-drying proteins

 The present invention relates to a method for freeze-drying a protein-containing solution, and more particularly to a method for freeze-drying an L-methionine arylase-containing solution. Background art

 A method for preserving a protein used as a medicine in a stable state for a long time has been long desired, and the freeze-drying method is one of the effective means. As a method for subjecting a solution containing a protein to freeze-drying, for example, Japanese Patent Application Laid-Open No. 7-227282 is known. In the patent application, L-methionine-lyase-containing solution contains (1) an oligosaccharide, (2) a water-soluble polysaccharide, (3) a sugar alcohol, (4) a protein, (5) an acidic amino acid, (6) One or more members selected from the group consisting of gluyuthione and (7) N-acetyl cysteine coexist, and freeze-drying prevents long-term reduction of L-methionine lyase activity. A method for doing so is disclosed. However, in this patent application, there is no report on suppression of a multimer formed during freeze-drying, and the organic acid metal salt used in the present invention is not included as an additive.

On the other hand, in International Publication No. WO 94/22471, a liquid preparation containing antithrombin-III is mixed with sodium citrate or the like as a stabilizer to prevent polymerization of antithrombin-III. A method for stably storing for a long time is disclosed. In Japanese Patent Application Laid-Open No. 9-11325354, dalconic acid or a salt thereof is added as a stabilizer to a liquid preparation containing antithrombin-III to stably store antithrombin-I II for a long period of time. A method is disclosed. However, these patents The application does not suggest anything about the suppression of the formation of protein multimers formed during lyophilization. Disclosure of the invention

 Purified L-methionine arylase and L-asparaginase are usually present in the solution in tetrameric form. When a solution containing such a protein, especially a solution containing L-methionine lyase, is lyophilized, and the lyophilized product is dissolved in a buffer or the like, the tetramer associates to form a multimer of 8 or more. Is formed. An object of the present invention is to suppress the formation of a polymer by associating a stable and biologically active minimum unit protein during lyophilization.

The formation of the multimer needs to be suppressed for the following reasons. One possibility is that the higher the molecular weight of the multimer formed, the higher the antigenicity may be. In other words, there is a possibility that the pharmaceutical protein itself may become an allergen, and it is necessary to prevent this. The second reason is that high quality preservation is required for drugs used as pharmaceuticals, and it is desirable that the quality before and after freeze-drying be constant.Before and after freeze-drying, there is a difference in quality due to the formation of multimers. Is not preferred. Therefore, it is necessary to suppress the formation of multimers. However, it has recently been the case that pharmaceutical protein preparations have been commercialized, and there have been few attempts to suppress the formation of these multimers. The present invention provides a method for easily producing a preparation containing a high-purity protein in which formation of a multimer is suppressed even when freeze-dried, and a method for freeze-drying a protein-containing solution. Is provided. The present inventors have conducted intensive studies to achieve the above object, and as a result, in a method for freeze-drying a protein-containing solution, particularly a L-methionine r-lyase-containing solution, the metal salt of an organic acid was added to the solution before freeze-drying. The present inventors have found that the addition of a salt can suppress the formation of a multimer of octamer or more protein even after lyophilization and subsequent dissolution, thus completing the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention provides a method for freeze-drying a solution containing a stable and biologically active minimum unit of a protein and a metal salt of an organic acid, and suppressing the protein from associating during lyophilization to form a multimer. A method for producing a freeze-dried product containing the protein; a method for producing a freeze-dried product of the present invention, wherein the protein is any of L-methionine lyase, L-asparaginase, and interleukin-2;

A method for producing the freeze-dried product of the present invention, wherein the smallest protein that is stable and biologically active is a tetrameric L-methionine-lyase, and the multimer is an 8-mer or more multimer;

The method of the present invention, wherein the organic acid is citric acid, acetic acid, lactic acid or dalconic acid; the method of the present invention, wherein the metal salt of the organic acid is sodium citrate, sodium acetate, sodium lactate or sodium gluconate;

The method of the present invention, wherein the concentration of the organic acid metal salt is 1 to 500 mM;

The method of the present invention, wherein the concentration of the organic acid metal salt is 50 to 200 mM;

A solid pharmaceutical composition comprising a lyophilized product produced by the method of the present invention;

A solid pharmaceutical composition comprising a lyophilized product containing a stable and biologically active minimum unit protein that forms a multimer during lyophilization and a metal salt of an organic acid that suppresses the formation of the multimer;

 A solid pharmaceutical composition of the present invention comprising a freeze-dried product containing L-methionine arylase and a metal salt of an organic acid;

A formulation using the pharmaceutical composition of the present invention; and

A method comprising suppressing freeze-drying of the protein to form a multimer by lyophilizing a solution containing a stable and biologically active minimum unit of a protein and an organic acid metal salt. About.

The “protein” according to the present invention includes proteins such as L-methionine lyase, L-asparaginase, and interleukin-2. L —methionine r-lyase is an enzyme that uses pyridoxal phosphate (hereinafter abbreviated as PLP) as a coenzyme [Tanaka, H., et al., Biochemistry 16, 100-106 (1977)]. W09 4/1 1 5 3 5 published May 26, 1994].

 Proteins may not be stable in monomers depending on their type. For example, in the case of L-methionine lyase and L-asparaginase, tetramers composed of the same subunit are stable and have biological activity. Thus, "stable and biologically active minimum unit protein" means a tetramer in L-methionine lyase and L-asparaginase. On the other hand, interleukin_2 is a monomer and has stability. Therefore, in the case of interleukin-2, "stable and biologically active minimum unit" means a monomer.

 The term “multimer” refers to a multimer associated with the aforementioned “stable and biologically active minimum unit protein”. Therefore, when the “protein” is L-methionine lyase or L-asparaginase, “multimer” means an octamer or more multimer. In the case of interleukin-2, "multimer" means a dimer or higher multimer.

 The protein concentration before lyophilization, especially the concentration of L-methionine-lyase, is in the range of 1-4 Omg / m 1. Since L-methionine lyase is actually used at about 30 mg / ml, it is preferably in the range of 20 to 40 mgZml.

 The present invention suppresses the formation of multimers by adding an organic acid metal salt as an additive. "Organic acids" include cunic, dalconic, lactic and acetic acids. The “metal” includes sodium, potassium, calcium, and the like. Preferably, sodium is used.

The concentration of the organic acid metal salt when the organic acid metal salt is added as an additive is preferably 1 to 50 OmM. However, considering the use of excipients, Preferably, it is added at 50 to 200 mM.

 The solution according to the present invention includes a buffer solution. In the examples of the present invention, a buffer solution of sodium phosphate (10 mM Na-phosphate buffer, pH 7.2) is used, but another buffer solution may be a buffer solution of a metal salt of citric acid. The solution containing L-methionine arylase preferably uses a sodium phosphate buffer, and the buffer preferably further contains PLP. Since L-methionine τ-lyase is unstable under weakly acidic conditions, the pH of the solution is preferably in the range of 6.5 to 9.0, but the particularly preferred ρ Η is 7.0 to 8.0. It is 0.

The term “at the time of freeze-drying” includes the time from the time when the protein-containing solution is freeze-dried using a freeze dryer to the time when the obtained freeze-dried product is dissolved with a buffer or the like.

 The protein-containing solution to which the organic acid metal salt has been added can be freeze-dried by an ordinary method. In Examples, after pre-freezing, freeze-drying was performed for 20 hours using a freeze dryer.

 The obtained freeze-dried product can be stored at an appropriate temperature, but a lower storage temperature allows a longer storage period, so that it is usually −80 to 30 ° C., preferably −40 to 1 ° C. It is desirable to store at 0 ° C.

 “Solid pharmaceutical composition” means a composition before dissolution including a freeze-dried product of a protein having a pharmaceutical use and a metal salt of an organic acid.

 The lyophilized protein obtained by the method of the present invention was dissolved in a buffer containing PLP (lOmM Na-phosphate buffer pH 7.2) in the Examples, but it was dissolved in other appropriate buffer or water ( Water for injection).

 "Preparation" means a liquid preparation, particularly an injection. Example

Hereinafter, the present invention will be described more specifically with reference to Examples and Reference Examples, but these do not limit the scope of the present invention. Reference Example 1: Purification of L-meteonin lyase

 1. Purification of L-methionine arylase

 According to the method described in Inoue, H. et al., J. Biochem. 117, 1120-1125 (1995), an expression plasmid incorporating the structural gene portion of L-methionine arylase was introduced into Escherichia coli, and A strain for expressing methionine arylase is prepared, and the strain is cultured. After culturing, the wet cells are disrupted, and an aqueous solution of polyethyleneimine is added to adjust the final concentration to 0.1-0.2% (w / v) .Then, the cells are treated at 5-25 ° C for 1-20 minutes. The residue is removed to obtain a crude enzyme solution of L_methionine r-lyase. Then, the obtained crude enzyme solution is salted out with ammonium sulfate and centrifuged, and the obtained precipitate is dissolved in a phosphate buffer to obtain an unpurified L-methionine 7 "-lyase solution. Polyethylene glycol at a final concentration of about 8 to 12% (W / V) is added to the unpurified L-methionine τ-lyase solution, and the mixture is stirred at 4 ° C for about 60 minutes, followed by centrifugation. 2. Production of L-methionine arylase crystals

 After removing the contaminants in step 1 above, the solution is heated and polyethylene glycol is added by an ordinary method. Subsequently, columnar crystals of L-methionine r-lyase are obtained by adding an inorganic salt. The L-methionine arylase crystals thus precipitated can be collected, for example, using a centrifuge. Reference Example 2: Measurement of multimers

 Using Size Exclusion HPLC (SE-HPLC), the state of formation of multimers before and after lyophilization was measured. SE-HPLC was performed under the following conditions.

 • Column: TSKgel G3000SW

 · Eluent: 10mM Na-phosphate buffer, pH7.2 (120mM NaCl)

-Flow rate: 1.0ml / min • Flow rate: l.Oml / min

• Detection: 220 nm Example 1: Addition of an additive to a solution containing L-methionine arylase (1) The crystals of L-methionine arylase obtained in Reference Example 1 were purified using a 100 MP LP (pyridoxalline) solution. Acid) and adjusted so that the concentration of L-methionine lyase was 30 mg / m 1 (at this point, L-methionine lyase was used). —The tetramer of lyase is 98.2%, whereas the multimer of 8 or more is 1.8%). To investigate the effect of buffer pH on multimer formation, two experimental systems were prepared, pH 7.2 and 8.0 of the buffer. The additives shown in Table 1 were separately prepared and added, and after pre-freezing, freeze-dried in a freeze dryer for 20 hours.

The lyophilized product was then dissolved in a buffer (10 mM Na-phosphate buffer, pH 7.2) containing PLP10-iM, and the formation of multimers of L-methionine lyase was measured by the method of Reference Example 2. . The results are shown in Table 1.

Multimer formation after freeze-drying for 30 mg / ml L-methionine arylase.

表 1に示すとおり、 3 0mgZm l の L—メチォニン ァ—リァ一ゼを凍結乾燥 させ、それを溶解後に L一メチォニン アーリァ一ゼの多量体の形成を参考例 2の 方法により測定した。 無添加では、 多量体は 22.6%であったのに対して、 ダルコ ン酸ナトリゥム添加では 1.7〜3.3%、 クェン酸ナトリゥム添加では 2.6〜5.5%で あった。 これら有機酸金属塩は、 添加物の P Hや添加物濃度にかからわず多量体 の形成を有意に抑制した。 さらに、 添加物の濃度が lOOmMの場合には、 酢酸ナ トリウム添加では、 多量体は 2.7〜4.2%、 乳酸ナトリウム添加では 2.0〜2.3%で あった。 これら有機酸金属塩も、 添加物の P Hにかかわらず無添加に比べて明ら かに多量体の形成を抑制した。 実施例 2 ： L—メチォニン アーリァーゼを含有する溶液への添加物の添加（2) L一メチォニン アーリァーゼの濃度が多量体形成に及ぼす影響を調べるため、 実施例 1ではし一メチォニン _r—リァーゼの濃度が 3 O mg/m 1であるのに 対して、 実施例 2ではその濃度を 1 mg/m 1 に調製して行なった。

As shown in Table 1, 30 mgZm L of L-methionine enzyme was lyophilized, and after dissolving, the formation of multimers of L-methionine enzyme was measured by the method of Reference Example 2. Without addition, the multimer content was 22.6%, whereas the addition of sodium dalconate was 1.7-3.3%, and that of sodium citrate was 2.6-5.5%. These organic metal salts significantly inhibited the formation of multimers regardless of the pH of the additive and the concentration of the additive. Furthermore, when the additive concentration was 100 mM, the amount of the multimer was 2.7 to 4.2% when sodium acetate was added, and 2.0 to 2.3% when sodium lactate was added. These organic acid metal salts also clearly inhibited the formation of multimers, irrespective of the pH of the additive, as compared to the case without the additive. Example 2: Addition of an additive to a solution containing L-methionine lyase (2) To examine the effect of the concentration of L-methionine lyase on multimer formation, in Example 1, the concentration of lymethionine _r -lyase was examined. Is 3 O mg / m 1 On the other hand, in Example 2, the concentration was adjusted to 1 mg / m 1.

 The crystals of L-methionine lyase obtained in Reference Example 1 were dissolved in a buffer (10 mM Na-phosphate buffer, pH 7.2) containing PLP10 OM, and the concentration of L-methionine γ-lyase was 1%. It was adjusted to be mgZm 1 (at this point, the tetramer of L-methionine arylase is 98.2%, whereas the multimer of 8 or more is 1.8%). In order to examine the effect of the pH of the buffer on multimer formation as in Example 1, two types of experimental systems, pH of the buffer 7.2 and 8.0, were prepared. The additives shown in Table 2 were separately prepared and then added. After pre-freezing, freeze-drying was performed for 20 hours using a freeze dryer.

 The lyophilized product was then dissolved in a buffer containing PLP10OiM (10 mM Na-phosphate buffer pH 7.2), and the formation of multimers of L-methionine lyase was determined by the method of Reference Example 2. It was measured. The results are shown in Table 2. Table 2

formation of multimers after lyophilization for L Mechionin _r Riaze of lmg / ml

1 mg / m 1 of the methionine lyase was lyophilized, and after dissolving, the formation of a multimer of L-methionine lyase was measured by the method of Reference Example 2. With no addition, the multimer was 16.1%, whereas with sodium dalconate, Was 3.2-4.6%. These organic metal salts significantly inhibited the formation of multimers regardless of the pH of the additive and the concentration of the additive. Industrial applicability

 An object of the present invention is to freeze-dry a solution containing a minimum unit of a stable and biologically active protein and a metal salt of an organic acid, and to suppress the formation of a multimer associated with the protein during freeze-drying. A method for producing the freeze-dried product containing the protein. According to the present invention, it is possible to provide a highly pure drug in which the formation of multimers is suppressed even during freeze-drying. In particular, the present invention provides a production method useful for formulating a drug containing L-methionine lyase.

Claims

The scope of the claims 1. It is characterized in that a solution containing a stable and biologically active minimum unit of a protein and an organic acid metal salt is freeze-dried, and the protein is prevented from associating during lyophilization to form a multimer. A method for producing the freeze-dried product containing the protein.  2. The method for producing a freeze-dried product according to claim 1, wherein the protein is any one of L-methionine arylase, L-asparaginase, and interleukin-1.  3. The stable or biologically active minimum unit protein is a tetramer L-methionine arylase, and the multimer is an octamer or higher multimer. For producing freeze-dried products.  4. The method according to claim 1, wherein the organic acid is citric acid, acetic acid, lactic acid or dalconic acid.  5. The method according to any one of claims 1 to 4, wherein the organic acid metal salt is sodium citrate, sodium acetate, sodium lactate or sodium dalconate.  6. The method according to any one of claims 1 to 5, wherein the concentration of the organic acid metal salt is 1 to 500 mM.  7. The method according to any one of claims 1 to 6, wherein the concentration of the metal salt of the organic acid is 50 to 200 mM.  8. A solid pharmaceutical composition comprising a freeze-dried product produced by the method according to any one of claims 1 to 7.  9. A solid pharmaceutical composition containing a freeze-dried product containing a stable and biologically active minimum unit protein that forms a multimer during lyophilization and a metal salt of an organic acid that suppresses the formation of the multimer .  10. The solid pharmaceutical composition according to claim 9, comprising a lyophilized product containing 10 .L-methionine r-lyase and a metal salt of an organic acid. 11. A preparation using the pharmaceutical composition according to claim 9 or 10. 12. A solution containing a minimum unit of a stable and biologically active protein and a metal salt of an organic acid is lyophilized, wherein the protein associates during lyophilization to form a multimer. How to suppress.