JP2002101879A - Useful protein composition and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition capable of stably preserving the physiological activity of the useful protein therein, and to provide a method for producing the composition. SOLUTION: This composition is obtained by adding at least two components selected from organic acids and alkali metal salts thereof, compounds having thiol structure and gum arabic, each being nonvolatile even if the composition undergoes a lyophilization treatment, to a useful protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a composition capable of stably retaining the physiological activity of a useful protein and a method for producing a stable useful protein composition.

[0002]

2. Description of the Related Art Proteins, especially enzymes, useful proteins having physiological activities, etc. can be mass-produced at low cost by genetic recombination technology and so on, and are used in various fields, especially in the fields of medicines, diagnostics and foods. It is supposed to be. On the other hand, proteins have the problem that their higher-order structure is easily destroyed by various external factors (temperature, light, pH, oxidation) during storage, and their functions (physiological activities) are lost. Methods for protecting the protein from these external factors and maintaining its biological activity have been studied.

At present, a useful and common method for stabilizing a protein is to mix it with another protein (gelatin, albumin, serum, collagen, etc.), and to mix it with gelatin or serum. Many enzymes and bioactive proteins that can be stored for a long period of time and have been commercialized as pharmaceuticals are known (Japanese Patent Laid-Open No.
-264728, JP-A-2-49734,
JP-A-54-80406, JP-A-56-6860
No. 7).

[0004] The stabilization of interferon (IFN), which is a physiologically active substance having an immunomodulatory action and an antiviral action and attracting attention in pharmaceutical applications, can be mentioned in JP-A-60-228422 and JP-A-60-228. No. 34919, JP-A-61-137828, JP-A-60-1987
No. 260523 discloses a method of stabilizing by mixing with albumin or gelatin. Examples of compounds other than proteins that have a protein stabilizing effect include methods of mixing with saccharides, particularly monosaccharides, disaccharides, and polysaccharides such as dextran and hydroxyethyl starch (JP-A-59-181223).
JP, JP-B-6-51641, JP-A-61-4
No. 4826, JP-A-60-155136) or a method using cyclodextrin or polysaccharide alcohol as a stabilizer (JP-A-58-92691, JP-B-3-500882).

[0005] There is also a method of predominating a dimer of IFN-γ having high activity by using lactobionic acid (Japanese Patent Publication No. 3-501604).

As a report of a mixture of gum arabic and protein, there are those using an aqueous solution of gum arabic as a dispersant for a sustained-release agent and those using it as a stabilizer in tablets (JP-A-6-321803, WO 985132).
8). Further, an example in which a useful protein is stabilized by mixing it with an aqueous solution of gum arabic (Japanese Patent Application No. 10-367).
567), but there is no known useful protein composition that is stable during long-term storage or storage under severe conditions and that suppresses generation of insoluble fine particles during storage.

[0007]

As a method for stabilizing a useful protein, it is known that it can be improved by adding another protein, a saccharide or a polyhydric alcohol in an aqueous solution. However, even in the lyophilized state, a decrease in physiological activity may be observed. Therefore, stable storage methods and compositions of useful proteins that do not decrease in physiological activity even under long-term storage or storage under severe conditions are desired. In addition, when a useful protein is used as a drug for injection, the formation of insoluble fine particles when used as an aqueous solution poses a problem. Therefore, a composition that does not produce insoluble fine particles even when reconstituted into an aqueous solution after lyophilization is desired.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have newly developed an organic acid buffer which does not volatilize its components by freeze-drying treatment represented by citrate buffer, and a thiol structure represented by cysteine. It has been found that by mixing an aqueous solution containing a compound having at least two of gum arabic and a gum arabic with a useful protein and freeze-drying it, the activity of the protein can be stably maintained even under severe conditions.
In addition, this composition can maintain physiological activity at a high level for a certain period of time in an aqueous solution state before freeze-drying, and when dissolved in an aqueous solution state after freeze-drying, the p.
When H is 7 or less, it has been found that the physiological activity can be maintained at a high level for a certain period, and that no insoluble fine particles are generated in an aqueous solution state, and the present invention has been achieved.

That is, the present invention provides a useful protein composition containing at least two of an organic acid and an alkali metal salt of an organic acid which are not volatilized in lyophilization, a compound having a thiol structure and gum arabic, and a pharmaceutical composition for injection containing the same. Acid buffer that does not volatilize its components by lyophilization in an aqueous solution containing a substance and a useful protein, a compound having a thiol structure, and gum arabic.
A method for producing a useful protein composition, comprising adding an aqueous solution containing the two components.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The useful protein of the present invention is not particularly limited, but includes organic acids, alkali metal salts of organic acids,
A compound having a thiol structure and a protein whose activity is not inhibited by gum arabic may be used, and a protein having an enzyme or a physiological activity, for example, interferon,
Examples include interleukins, insulin, growth hormone, and the like. The useful proteins are preferably cytokines, and more preferably interferon-α, β, γ or ω. The interferon is preferably a vertebrate interferon, and more preferably an animal-derived interferon such as canine interferon (α, β, and γ types), feline interferon ω, and the like.

According to the present invention, at least two selected from (1) organic acids and alkali metal salts of organic acids which are not volatilized in freeze-drying, (2) compounds having a thiol structure, and (3) gum arabic. is important. Specifically, (1) an organic acid and an organic acid alkali metal salt that does not volatilize during lyophilization, and (2) when containing a compound having a thiol structure, (2) a compound having a thiol structure,
And (3) when containing gum arabic, (1) an organic acid and an organic acid alkali metal salt that does not evaporate in freeze-drying, and (3) may contain gum arabic, preferably,
(1) An organic acid and an organic acid alkali metal salt that does not volatilize in freeze-drying, and (2) a compound containing a compound having a thiol structure, more preferably (1) an organic acid and an organic acid that does not volatilize in freeze-drying Alkali metal salts,
(2) a compound having a thiol structure; and (3) gum arabic.

In the present invention, an organic acid and an organic acid alkali metal salt used for improving the storage stability of a protein may have, as a secondary component, a component that has a pH buffering ability and deactivates a protein when combined. not included,
All organic acids and organic acid alkali metal salts that do not volatilize by lyophilization, including citric acid, sodium citrate, succinic acid, sodium succinate, tartaric acid, sodium tartarate, malic acid, and sodium malate Salt and the like. Preferably, citric acid or sodium citrate is used.

In the present invention, the compound having a thiol structure used for improving the storage stability of a protein is defined as a component which inactivates the protein or a component which prevents the protective action of the protein by the thiol structure as an auxiliary component. And all compounds having a thiol structure, not including, such as sodium thiosulfate, sodium thioglycolate, thioglycerol, and glutathione. Preferably, cysteine is used.

The present inventors have searched for a composition that can maintain the activity of interferon stably even under severe conditions during storage. In particular, the inventors have focused on the inactivation of physiological activity and pH change due to an oxidation reaction during storage. Cysteine is selected from compounds having a thiol structure known to have an antioxidant effect, and citrate buffer is selected from non-volatile organic acid buffers. Examination of a composition comprising an acid buffer, cysteine and gum arabic has been found to improve storage stability as expected. It can easily be assumed that this storage stability improving effect can be obtained also in other compounds having a thiol structure which is also considered to have an antioxidant effect, and can also be obtained in other nonvolatile organic acid buffers. This can be easily assumed.

The concentration of the organic acid or the alkali metal salt of the organic acid in the aqueous solution may be any concentration.
The concentration of 5 mM or more in a solution state is preferable because the H buffering capacity is reduced, and more preferably 10 to 100 m
M.

The concentration of the compound having a thiol structure in the aqueous solution may be any concentration. However, if the concentration is too low, the effect of improving storage stability is small, and if the concentration is high, insoluble fine particles may be generated. From 2 to 8 mM in a solution state, more preferably 4 to 6 mM.
mM.

The concentration of gum arabic may be any concentration, but if the concentration is too low, the effect of improving storage stability is small, and if the concentration is high, the viscosity increases and it is difficult to handle.
The concentration is preferably from 0.01 to 10.0% (W / V), more preferably from 0.5 to 2.0% (W / V).

Useful proteins can be obtained by temperature changes due to freezing, thawing, heating, etc., extraction, purification, and lysis in buffer.
It is easy to lose its physiological activity due to external factors such as H change, but prior to the operation to induce these inactivations, it is mixed with an organic acid buffer which does not volatilize by freeze-drying and an aqueous solution containing a compound having a thiol structure. By doing so, its biological activity is significantly maintained.

In general, it is known that in a protein purification operation, when the purification step proceeds and the purity of the target protein increases, the protein is easily deactivated. In other words, the purification operation is also one of the operations that induces inactivation, but by mixing with an organic acid buffer that does not volatilize by lyophilization and a compound having a thiol structure during or before the purification. In addition, protein inactivation due to purification can be prevented.

The inactivation of useful proteins with time in an aqueous solution, that is, the inactivation during storage, is prevented by mixing with an aqueous solution of an organic acid buffer, a compound having a thiol structure, and a gum arabic which does not volatilize by lyophilization. It is possible. An aqueous solution containing an organic acid buffer that does not volatilize by freeze-drying, a compound having a thiol structure, and gum arabic is preferably 15 ° C. or lower, depending on the thermal stability of a useful protein when stored in a liquid state. If the mixture does not freeze, the lower the temperature, the better, more preferably 4 to 10C.

When the storage is to be carried out for a long period of time, it is preferable to freeze or freeze-dry the composition for storage. In the case of cryopreservation, the temperature may be any temperature as long as the temperature is low enough not to be thawed.

In the case of freeze-drying, since the preservability increases as the water content decreases, it is preferable to dry the water to 5% by weight or less. The composition thus freeze-dried is preferably stored in a cool, dark place, and can be stably stored for one year if stored at room temperature or longer if stored refrigerated. In addition, even under the severe condition of 50 ° C., storage stability for one month or more can be expected. The freeze-dried product is used after being dissolved again in a solution such as water or physiological saline, and the storage after the re-dissolution is the same as the storage method in the liquid state.

The preferred pH for storing useful proteins in solution depends on the pH stability of the useful proteins themselves. However, by adding an organic acid buffer, a compound having a thiol structure and a gum arabic that does not volatilize by lyophilization to a useful protein solution, the pH range in which the useful protein can be stably stored is widened. For example, interferon-γ used in the present invention can maintain a high activity of 80% or more in a pH range of 5.5 to 8 by adding citric acid, sodium citrate, cysteine and gum arabic to an aqueous solution thereof.

The preferred pH for long-term storage or storage under severe conditions of a freeze-dried product of a useful protein depends on the pH stability of the useful protein itself. However, long-term storage of useful proteins or storage under severe conditions by mixing useful proteins with at least two of organic acids and organic acid alkali metal salts which do not volatilize during freeze-drying, compounds having a thiol structure and gum arabic Can maintain stability in a wide range of pH. For example, the interferon-γ used in the present invention is stably stored under severe conditions in the pH range of 5.5 to 6.7 by freeze-drying after adding citric acid, sodium citrate, cysteine and gum arabic. I was able to. When the pH exceeded 7, a slight decrease in the residual activity was observed.

Mixtures of useful proteins with at least two of organic acids and organic acid alkali metal salts which do not volatilize in the freeze-drying process, compounds having a thiol structure, and gum arabic are used for various purposes based on the functions of the proteins. obtain. If the useful protein is of a grade that is useful for pharmaceutical use and has no problem for pharmaceutical use, organic acids, alkali metal salts of organic acids, compounds having a thiol structure, and gum arabic are listed on the Japanese Pharmacopoeia or added to pharmaceutical grades. By doing so, the mixture can be used for pharmaceutical purposes.

In addition, the enzymes used for various purposes of measurement and diagnosis other than pharmaceutical use can also be used for the method of improving the storage stability disclosed by the present invention, and the storage stability can be improved. Can be expected to be improved, and long-term use becomes possible.

A useful protein composition containing at least two of an organic acid and an organic acid alkali metal salt which does not volatilize in the freeze-drying process, a compound having a thiol structure, and gum arabic does not inhibit the activity of the protein other than these components. Any compound can be included. For example, polysaccharides other than gum arabic, polyols including polyethylene glycol, polyoxyethylene hydrogenated castor oil, surfactants such as Tween 20, sugars such as sorbitol, amino acids such as glycine, and gelatin such as Protein may be added.
Preferably, a surfactant is added. As the surfactant, at least one selected from polyoxyethylene hydrogenated castor oil and polyoxyethylene sorbitan fatty acid ester
Preferably it is a seed.

Since there is no problem even if salt is added,
For example, when the composition is used as a medicament for injection, the osmotic pressure can be adjusted with sodium chloride. In particular, in order to stably store a useful protein at a low concentration in an aqueous solution or as a lyophilized product, at least two components of an organic acid and an organic acid alkali metal salt, a compound having a thiol structure, and a gum arabic which do not volatilize during the lyophilization treatment. In addition, it is useful to further add a surfactant.

When a useful protein is used as a medicament for injection, insoluble fine particles must not be formed in an aqueous solution thereof.
It is assumed that the production of insoluble fine particles is suppressed by the following. For example, the interferon-γ used in the present invention was mixed with citric acid, sodium citrate, cysteine, gum arabic, polyethylene glycol, and polyoxyethylene hydrogenated castor oil to prepare various pH values. After freeze-drying, the composition was reconstituted into an aqueous solution. In the case of a composition having a pH of more than 7, formation of insoluble foreign matter was observed.
When H was 7 or less, no insoluble foreign matter was generated. . Further, interferon-γ is mixed with citric acid, sodium citrate, cysteine, gum arabic, polyethylene glycol, and polyoxyethylene hydrogenated castor oil, and the pH is adjusted.
After adjusting to 5.5 and freeze-drying, the freeze-dried product was dissolved in physiological saline and restored to the original concentration.
No generation of insoluble fine particles was observed even when stored at ℃ for up to 3 weeks, and no generation of insoluble fine particles was observed when the freeze-dried product was stored under severe conditions at 50 ° C. for one week and subjected to the same test. .

As described above, the useful protein composition and the method for producing the useful protein composition of the present invention can be used for freeze-drying a useful protein aqueous solution to prepare a formulation and then re-dissolving it with water, for example, for injection. It is useful in the production of pharmaceuticals, diagnostics, and the like.

The method for producing a useful protein in the present invention is not limited, and may be any method such as a protein produced using an organic synthesis method, a recombinant DNA technique or a protein produced by extraction and purification from natural products. Applicable to produced proteins. For example, it can be preferably applied to stabilization of useful proteins such as canine interferon-γ and feline interferon ω produced by Escherichia coli or silkworm.

[0032]

The present invention will be specifically described below with reference to examples. The compounds having a citric acid, sodium citrate salt and thiol structure used in the examples were of a pharmaceutical grade or a pharmaceutical additive grade.

Reference Example <Preparation of canine interferon-γ by silkworm> A virus solution of the recombinant baculovirus rBNVγ disclosed in Japanese Patent Application No. 9-208087 was inoculated into silkworm larvae of 5 days and 2 days, and incubated at 25 ° C. for 4 hours. The animals were bred on a commercial artificial feed (Kanebo Silk Elegance) for one day. The abdomen of 50 silkworms was cut, and 500 ml of 50 mM acetate buffer (pH 3.0) containing 0.01% benzalkonium chloride was used.
5) and kept at 4 ° C. for 20 hours. The resulting silkworm body fluid extract was neutralized with 2N NaOH,
The mixture was centrifuged at 0 rpm for 15 minutes, and the supernatant was collected. The obtained supernatant is subjected to a hollow fiber type ultrafiltration membrane device (Amic
on, manufactured by HIP40-10
Ultrafiltration was performed using 0). After passing the treatment solution through a copper chelate column, the solution is applied to a Q Sepharose column, washed with a 20 mM glycine buffer (pH 9.0),
The adsorbate is eluted with a linear concentration gradient of sodium chloride. A fraction containing canine IFN-γ was collected from the eluted fraction by reverse phase HPLC analysis, further applied to a Blue Sepharose column, and subjected to a 20 mM acetate buffer (pH 5.5).
After washing in 5), the adsorbate is eluted with a linear concentration gradient of sodium chloride. Reverse layer HPL from the eluted fraction
The fraction containing canine IFN-γ was collected by C analysis, and the fraction obtained by desalting the fraction was used as a canine IFN-γ sample for the study of the stabilizer.

<Method for measuring antiviral activity> The activity of canine interferon-γ is determined as an antiviral activity by the method of Prober et al., Science 238, 336-3.
41 (1987). Vesicular Stomatitis Virus was used as a virus for measurement, and dog MDCK (ATCC CCL-34) was used as a sensitive cell. That is, dog MDC cultured at 37 ° C. until confluence on a 96-well microplate.
A diluted solution of a sample containing canine IFN-γ was added to the K cells, and the mixture was further cultured at 37 ° C. for 20 to 24 hours to induce antiviral activity. Add VSV and add
After culturing for 20 hours, surviving dog MDCK cells adhering to the microplate were stained with a crystal violet stain containing 20% formalin. By measuring the amount of crystal violet on the microplate and measuring the absorbance at 570 nm, the amount of canine IFN-γ at which cells survived 50% was defined as 1 unit (1 U) of antiviral activity.

The standard deviation of the antiviral activity data obtained by this method is 32%.

<Measuring method of insoluble fine particles> After the lyophilized product is stored under various conditions, physiological saline is added without opening the package using a syringe for injection and a needle. After gently stirring and confirming that it is colorless and clear, it is further stored under various conditions.

A sample of 6 ml was placed in one sample tube, left at room temperature for 1 hour for degassing, and then subjected to a light shielding type automatic particle measuring device (8000A + 3000A,
(Hiac / Leuco). As a result of the measurement, a sample in which 6000 or more insoluble fine particles having a size of 10 μm or more and 600 or more insoluble fine particles having a size of 25 μm or more per 1 ml of the aqueous solution was regarded as having produced insoluble fine particles.

Example 1 A canine IFN-γ sample extracted and purified from a silkworm inoculated with a recombinant virus was applied to a final concentration of 20 mM acetate buffer (pH 5.5) or 20 mM citrate buffer (pH 5.5), respectively.
5.5) was added, and a group was made with or without the addition of cysteine at a final concentration of 4 mM. In addition to these compositions, gum arabic, polyethylene glycol, glycine, and polyoxyethylene hydrogenated castor oil were each 1% (W / V) in final concentration and 0.1%.
5% (W / V), 0.075% (W / V), 0.01% (W / V)
Lyophilized. After storing these at 50 ° C. for one week, the activity was measured. The activity retention rate was expressed assuming that the activity of the sample immediately after freeze-drying was 100%. Table 1 shows the results.

The freeze-dried product was redissolved in the same volume of physiological saline as before freeze-drying, and used for measurement of antiviral activity.

The residual activity was improved by adding cysteine to the acetate buffer. By changing the acetate buffer to the citrate buffer, no significant improvement was observed, but by adding cysteine to the citrate buffer, a remarkable improvement in the residual activity was observed.

[0041]

[Table 1]

Example 2 A canine IFN-γ sample extracted and purified from a silkworm inoculated with a recombinant virus was subjected to a citrate buffer (pH 5.
A mixture of 5) and an acetate buffer (pH 5.5) at the molar ratio shown in Table 2 was added to a final concentration of 20 mM. Further, cysteine was added at the final concentration shown in Table 2. After adding the final concentrations of gum arabic, polyethylene glycol, glycine and polyoxyethylene hydrogenated castor oil shown in Example 1 and freeze-drying, the residual activity after storage at 50 ° C. for 2 weeks was measured.
(The activity of the sample immediately after lyophilization is 6.8 × 10 ⁴ U / ml
Met. ) The results are shown in Table 2.

From Table 2, it can be seen that the improvement of the residual activity was attained by cysteine 2m
Although a slight increase was observed with the addition of M, a marked improvement was observed with the addition of 4 mM or more. In addition, the addition of the citrate buffer showed a remarkable improvement in the residual activity at a molar ratio of at least 3 times that of the acetate buffer.

[0044]

[Table 2]

Example 3 A canine IFN-γ sample (20 mM acetate buffer, pH 5.5) extracted and purified from silkworms inoculated with the recombinant virus
To a final concentration of 20 mM acetate buffer (pH 5.5) or a final concentration of 20 mM citrate buffer (pH 5.5, molar ratio to acetate buffer of 6) and a final concentration of 4 mM cysteine. Add. Further, gum arabic, polyethylene glycol, glycine, and polyoxyethylene hydrogenated castor oil were added in the same manner as in Example 1, and after freeze-drying, the residual activity over time at 50 ° C. storage (the activity at the start of storage was 100% Was examined.
Table 3 shows the results.

In the composition of the acetate buffer, a remarkable decrease in the residual activity under severe conditions was observed, but the addition of the citrate buffer and cysteine could suppress the decrease in the residual activity.

[0047]

[Table 3]

Example 4 A canine IFN-γ sample (20 mM acetate buffer, pH 5.5) extracted and purified from silkworms inoculated with the recombinant virus
Citrate buffer (pH 5.5, molar ratio to acetate buffer: 6) at a final concentration of 4 mM
Cysteine, 1% (W / V) gum arabic, 0.5% (W / V)
V) Polyethylene glycol, 0.075% (W / V) glycine and 0.01% (W / V) polyoxyethylene hydrogenated castor oil were added to adjust the pH shown in Table 4 using NaOH. did. Table 4 shows the residual activity and the residual activity ratio (the activity at the time of preparation was defined as 100%) after storing them at 4 ° C for 3 weeks.

When the solution was stored at 4 ° C. for 3 weeks, excellent solution stability could be obtained in the pH range of 5.5 to 8.

[0050]

[Table 4]

Example 5 A canine IFN-γ sample extracted and purified from a silkworm inoculated with a recombinant virus (20 mM acetate buffer, pH 5.5)
In a final concentration of 20 mM citrate buffer (molar ratio to acetate buffer: 6), 4 mM cysteine, 1% (W / V) gum arabic, 0.5% (W / V) ) Polyethylene glycol, 0.075% (W / V) glycine,
01% (W / V) polyoxyethylene hydrogenated castor oil was added using citric acid and NaOH to give p
After adjusting to H, it was freeze-dried. Table 5 shows the change in activity and the residual activity ratio when each of them was stored at 50 ° C. for 2 weeks (the activity immediately after freeze-drying was defined as 100%, respectively).

PH 5.5 for storage under severe conditions
From 7.4 to 7.4, a good activity retention rate was exhibited.
With H5.5 and 6.7, excellent storage stability could be obtained.

[0053]

[Table 5]

Example 6 A canine IFN-γ sample (20 mM acetate buffer, pH 5.5) extracted and purified from silkworms inoculated with the recombinant virus
Citrate buffer (pH 5.5, molar ratio to acetate buffer: 6) at a final concentration of 4 mM
Cysteine, 1% (W / V) gum arabic, 0.5% (W / V)
V) polyethylene glycol, 0.075% (W / V) glycine, 0.01% (W / V) polyoxyethylene hydrogenated castor oil were added, and the antioxidants shown in Table 6 were added to a final concentration of 4 mM. Added in. Table 6 shows the activity and the residual ratio of the activity (the activity immediately after lyophilization is defined as 100%) when these were freeze-dried and stored at 55 ° C. for 3 days.

In storage under severe conditions, no improvement was observed with ascorbic acid, but the residual activity was improved with the thiol compound as compared to no addition. Among them, cysteine was able to maintain almost 100% activity.

[0056]

[Table 6]

Example 7 A canine IFN-γ sample (20 mM acetate buffer, pH 5.5) extracted and purified from silkworms inoculated with the recombinant virus
Citrate buffer (pH 5.5, molar ratio to acetate buffer: 6) at a final concentration of 4 mM
Cysteine, 1% (W / V) gum arabic, 0.5% (W / V)
V) polyethylene glycol, 0.075% (W / V) glycine and 0.01% (W / V) polyoxyethylene hydrogenated castor oil were added using citric acid and NaOH.
And then freeze-dried. Table 7 shows the formation of insoluble foreign matter when they were redissolved.

In the range of pH 5.5 to 6.7, no formation of insoluble foreign matter was observed, but at pH 7 or higher, formation of insoluble foreign matter was observed.

[0059]

[Table 7]

Example 8 A canine IFN-γ sample (20 mM acetate buffer, pH 5.5) extracted and purified from silkworms inoculated with the recombinant virus
Citrate buffer (pH 5.5, molar ratio to acetate buffer: 6) at a final concentration of 4 mM
Cysteine, 1% (W / V) gum arabic, 0.5% (W / V)
V) polyethylene glycol, 0.075% (W / V) glycine and 0.01% (W / V) polyoxyethylene hydrogenated castor oil were lyophilized and lyophilized at 4 ° C or 50 ° C. Saved for a week. After that, the cells were dissolved in the original volume of physiological saline and stored at 5 ° C. or room temperature, respectively, and the number of insoluble fine particles in the solution was measured over time. Table 8 shows the results.

After the lyophilized product was dissolved and stored at 4 ° C. or at room temperature for 3 weeks, no insoluble fine particles were formed. Even after the lyophilized product was stored at 50 ° C. for one week, a similar test was performed, but no formation of insoluble fine particles was observed.

[0062]

[Table 8]

[0063]

According to the present invention, a stable useful protein composition such as interferon is produced by adding at least two of an organic acid and an organic acid alkali metal salt which are not volatilized in the freeze-drying process, a compound having a thiol structure and gum arabic. can do.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 47/02 A61K 47/20 47/12 47/32 47/18 47/36 47/20 47/46 47 / 32 A61P 31/12 47/36 37/02 47/46 43/00 117 A61P 31/12 C07K 14/57 37/02 C12P 21/02 F 43/00 117 C12N 15/00 A C07K 14/57 A61K 37 / 02 C12P 21/02 37/66 HF term (reference) 4B024 AA01 BA25 CA04 EA02 HA01 4B064 AG12 CA10 CA12 CA19 CC24 CD07 CD13 CD30 CE11 CE12 DA01 4C076 AA12 AA29 BB11 CC29 CC35 DD01 DD23 DD43 DD51 EE23 EE30 FF14 GG36 FF63 GG46 4C084 AA03 AA06 BA44 CA17 DA24 MA05 MA17 MA44 MA66 NA01 NA02 NA03 ZB072 ZB332 ZC611 ZC612 4H045 AA10 AA20 BA10 CA40 DA18 EA20 FA72 FA74 GA23 GA25 GA26

Claims (22)

[Claims] 1. A useful protein composition comprising at least two selected from (1) organic acids and alkali metal salts of organic acids which are not volatilized in lyophilization, (2) compounds having a thiol structure, and (3) gum arabic. object. 2. An organic acid and an organic acid alkali metal salt which are not volatilized in freeze-drying, a compound having a thiol structure,
And a useful protein composition comprising gum arabic. 3. The useful protein composition according to claim 1, further comprising a surfactant. 4. The useful protein composition according to claim 1, wherein the organic acid and the organic acid alkali metal salt are citric acid and sodium citrate, respectively. 5. The useful protein composition according to claim 1, wherein the compound having a thiol structure is cysteine. 6. The method according to claim 1, wherein the lyophilized product is freeze-dried.
The useful protein composition according to any one of claims 1 to 5, wherein 7. The useful protein composition according to claim 6, wherein the composition is reconstituted into an aqueous solution after lyophilization. 8. The composition according to claim 6, wherein the pH is 7 or less when the aqueous solution is reconstituted after freeze-drying.
Useful protein composition as described. 9. The useful protein composition according to claim 6, wherein no production of insoluble fine particles is observed when the aqueous solution is restored to the original concentration after freeze-drying. 10. The useful protein composition according to any one of claims 1 to 9, wherein the useful protein is a cytokine. 11. The useful protein composition according to claim 1, wherein the cytokine is canine interferon-γ. 12. The method according to claim 1, further comprising at least one of polyethylene glycol, glycine and salt.
The useful protein composition according to any one of claims 1 to 7. 13. An injectable pharmaceutical composition comprising the useful protein composition according to any one of claims 1 to 12. 14. An aqueous solution containing a useful protein, (1) an organic acid buffer in which the components are not volatilized by freeze-drying,
A method for producing a useful protein composition, comprising mixing with an aqueous solution containing at least two of (2) a compound having a thiol structure and (3) gum arabic. 15. A useful protein composition characterized by mixing an aqueous solution containing a useful protein with an aqueous solution containing an organic acid buffer, a compound having a thiol structure and a gum arabic, the components of which are not volatilized by freeze-drying. Production method. 16. An organic acid buffer wherein the hydrogen ion concentration is pH
The method for producing a useful protein composition according to claim 14 or 15, wherein the value is 5.5 to 8.0. 17. The method for producing a useful protein composition according to claim 14, wherein the organic acid buffer is a citrate buffer. 18. The method for producing a useful protein composition according to any one of claims 14 to 17, wherein the compound having a thiol structure is cysteine. 19. The method for producing a useful protein composition according to claim 18, wherein cysteine is mixed at a concentration of 2 mM to 8 mM. 20. An aqueous solution containing a useful protein mixed with an aqueous solution containing at least two of an organic acid buffer, a compound having a thiol structure, and gum arabic, the components of which are not volatilized by freeze-drying, is freeze-dried. The method for producing a useful protein composition according to any one of claims 14 to 19, wherein 21. The method for producing a useful protein composition according to any one of claims 14 to 20, wherein the useful protein is a cytokine. 22. The method for producing a useful protein composition according to claim 14, wherein the cytokine is canine interferon-γ.