JP5990880B2 - Useful protein composition and method for producing the same - Google Patents

Description

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

  Proteins, especially enzymes, useful proteins with physiological activity, etc. can be mass-produced at low cost by genetic recombination technology, etc., so they are used in various fields, especially in the fields of medicine, diagnostics and food. ing. On the other hand, during storage, proteins have the problem that their higher-order structure is easily broken by various external factors (temperature, light, pH, oxidation) and lose their functions (physiological activity). Methods for protecting proteins from these external factors and maintaining physiological activity have been studied.

  Currently, it is useful and general as a protein stabilization method to be mixed with other proteins (gelatin, albumin, serum, collagen, etc.) and stored for a relatively long time by mixing with gelatin or serum. Many enzymes and bioactive proteins that have become available and have been commercialized as pharmaceuticals are known (Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4).

  If stabilization of interferon (IFN) which is a physiologically active substance having an immunomodulating action and an antiviral action and is attracting attention in pharmaceutical use, albumin is disclosed in Patent Document 5, Patent Document 6, Patent Document 7, and Patent Document 8. And a method of stabilizing by mixing with gelatin. Examples of compounds other than proteins that have a stabilizing effect on proteins include saccharides, particularly monosaccharides, disaccharides, and polysaccharides such as dextran and hydroxyethyl starch (Patent Document 9, Patent Document). 10, Patent Document 11, Patent Document 12) or a method using cyclodextrin or polyhydric sugar alcohol as a stabilizer (Patent Document 13, Patent Document 14).

  As a report of a mixture of a thiol compound and a protein, there is a method (Patent Document 15) for preventing deterioration of a desired aroma supply component and stabilizing food or beverage products by adding cysteine, and further, freezing. By adding at least two of organic acids and organic acid alkali metal salts that do not volatilize in the drying process, compounds having a thiol structure, and gum arabic to useful proteins, the generation of insoluble fine particles after dissolution is suppressed and stabilization is achieved. A useful protein composition and a method for producing the same have been clarified (Patent Document 16).

JP-A-2-264728 JP-A-2-49734 JP-A-54-80406 JP 56-68607 A JP 60-228422 A JP-A-60-34919 JP-A 61-137828 JP 60-260523 A JP 59-181223 A Japanese Patent Publication No. 6-51641 Japanese Patent Laid-Open No. 61-44826 JP 60-155136 A JP 58-92691 A Japanese Patent Publication No. 3-50082 JP-T-2004-518449 JP 2002-101879 A

  However, when a thiol compound is added for the purpose of stabilizing the useful protein composition by preventing oxidation, the thiol compound is oxidized by oxygen in the air and loses its antioxidant power. There is a problem in that a decrease in physiological activity of useful proteins is promoted by causing precipitation during storage due to poorly soluble substances. Therefore, there is a demand for a method and composition for preventing oxidation of the thiol compound and stably storing useful proteins even when a thiol compound is added.

  Therefore, this invention makes it a subject to aim at stabilization of useful protein by preventing the oxidation of the added thiol compound regarding the useful protein composition containing a thiol compound.

  As a result of intensive studies, the present inventors have reduced the dissolved oxygen concentration of a useful protein composition comprising a useful protein, a solvent, and a compound having a thiol structure, thereby preventing oxidation and precipitation of the thiol compound and stabilizing the protein activity. It was found that it can be maintained at the same, and the present invention was reached.

That is, the present invention comprises a useful protein composition or a useful protein composition characterized by comprising a useful protein, a solvent, and a compound having a thiol structure, and a dissolved oxygen concentration of 3 mg / L or less. a method for producing a compound of the choking gas into contact with a composition comprising and wherein the benzalkonium useful protein composition.

  A useful protein composition such as a stable interferon can be produced by removing oxygen from a composition containing a useful protein, a solvent, and a compound having a thiol structure.

1 is a graph showing changes with time in cysteine and cystine concentrations in Example 4. FIG. FIG. 2 is a graph showing the time course of antiviral activity in Example 4.

  The useful protein used in the present invention is not particularly limited as long as it is a protein whose activity is not inhibited by a compound having a thiol structure and a solvent. It is more desirable if it is a protein whose activity is not inhibited by the substance to be added, such as an acid or a salt thereof, a polysaccharide, a saccharide, an amino acid, a protein, or a nucleic acid that can be optionally added. Examples include interferon, interleukin, insulin, growth hormone, and the like. Useful proteins are preferably cytokines, and more preferably interferon-α, β, γ, or ω. Interferon is preferably a vertebrate interferon, and canine interferon (each type of α, β, γ), feline interferon ω, and the like, which are interferons derived from animals. Most preferred is canine interferon γ.

  In the present invention, the compound having a thiol structure used for improving the storage stability of a protein may be a compound having a thiol group, but preferably a component that inactivates a protein or a protein having a thiol structure. It is a compound having all thiol structures that does not contain a component that prevents the protective action as an accessory component, and includes cysteine, sodium thiosulfate, sodium thioglycolate, thioglycerol, glutathione, and the like. More preferably cysteine is used.

  The solvent used in the present invention may be any commonly used solvent, and any organic or inorganic solvent may be used as long as it does not denature a desired useful protein. Preferably, water or a buffer solution is used. The buffer solution may be any commonly used buffer, and preferred examples include a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, and a Tris buffer solution.

  Useful proteins are easily oxidized due to external factors such as temperature change and pH change, as well as the influence of oxygen in the air, and lose their physiological activity. However, by mixing with a solution containing a compound having a thiol structure, oxidation of useful proteins is suppressed, and the physiological activity is maintained significantly.

  However, a compound having a thiol structure is also easily oxidized by the influence of oxygen in the air, and when it becomes an oxidant, it loses its antioxidant ability and leads to oxidation of useful proteins. Furthermore, a thiol compound may produce a hardly soluble compound with oxidation. When a poorly soluble compound is produced, the compound easily precipitates, which leads to denaturation of useful proteins. In other words, in order to improve the stability of useful proteins, an appropriate amount of thiol compound is added to the useful protein, then oxygen present in the composition is removed, and the environment where the thiol compound and useful protein are not easily oxidized. It can be said that it is useful to create

  The present inventors have searched for a composition that can maintain interferon activity in a stable manner, and a method for producing the composition, and in particular, the disappearance of the antioxidant ability due to the oxidation reaction of the thiol compound contained in the composition or the poorly soluble thiol. Focusing on the occurrence of compound oxidant precipitation and the deactivation of interferon bioactivity associated with those phenomena, contacting the composition with a suffocating gas such as nitrogen, preferably removing the oxygen by carrying out a sufficient amount of bubbling, It was found that the oxidation reaction of thiol compounds was suppressed and the stability of interferon was improved.

  As a method for removing the oxygen, the dissolved oxygen is removed by bubbling other suffocating gas, preferably a non-oxidizing gas having no oxidizing power, or by degassing the composition under reduced pressure. Industrially available methods can be employed.

  When contacting the suffocating gas, the suffocating gas generally includes nitrogen, carbon dioxide, neon, argon, helium, xenon, chlorine, phosgene, etc. Among them, suffocation that preferably has no oxidizing power Sex gases such as nitrogen, carbon dioxide, neon, argon, helium, xenon and the like. Among these, nitrogen and argon are preferable, and nitrogen is most preferable.

  As a method for bubbling, a generally used method may be used, and examples thereof include a method in which a tube in which a suffocating gas flows is sufficiently ventilated while being introduced into the composition, preferably at the center bottom. In this case, in order to increase the efficiency of bubbling, piping or an air stone that can be ventilated over the entire composition can be installed, or the composition can be stirred while being uniformly aerated.

  The dissolved oxygen concentration in the composition when oxygen is removed by the above method should be controlled by the type of composition and the period of stable storage, but generally 3 mg / L or less is desirable. . It is preferable to keep the dissolved oxygen concentration lower when it is desired to stably store a useful protein that is more easily oxidized, or when a longer period of storage is desired. Preferably it is 2 mg / L or less. Although it is preferable that the lower limit is not detected, the useful protein can be stabilized over a considerably long period if it is about 0.01 mg / L.

  The concentration of the compound having a thiol structure in the solution may be any concentration, but if the concentration is too low, the effect of improving the storage stability is small, and if the concentration is high, there is a concern that precipitation is likely to occur. In the case of cysteine preferably used in the present invention, a concentration of 0.1 to 0.8 g / L is preferable. Also when using the compound which has another thiol structure, what is necessary is just the density | concentration in which the effective amount for obtaining desired stability is contained.

  When cysteine is used, the remaining proportion affects the antioxidant effect of the entire composition. Since cystine in which cysteine is oxidized does not have antioxidant capacity due to its structure, it is desirable that the cystine ratio is low. Therefore, the ratio of cystine is 50% by mass, preferably 40% by mass or less of the total amount of cysteine and cystine present at most.

  In the case of long-term storage, the composition is preferably stored frozen or lyophilized. The storage temperature for cryopreservation may be any number as long as the composition does not melt, and is the same as when stored in a liquid state after thawing.

  In the case of lyophilization, the smaller the amount of the remaining solution, the better the storage stability. Therefore, it is better to dry as much as possible. When water or a buffer solution is used as a solvent, it is preferable to dry it to a moisture content of 7% by weight or less. The composition thus freeze-dried can be stably stored for a long time under normal storage conditions. Preferably it is room temperature or a cool dark place storage (for example, 0-30 degreeC), More preferably, it is a cool dark place (for example, 0-10 degreeC). In particular, if it is stored in a cool and dark place, it can be stably stored for a period exceeding several years. Moreover, the preservation | save property of one month or more may be expected also under severe conditions of 50 degreeC. The freeze-dried product is redissolved with a solvent such as water, buffer solution, physiological saline, etc. and restored to a solution for use. However, the storage after redissolving is the same as the storage method in a liquid state. .

  The useful protein composition containing the compound having the above thiol structure can be used for various applications based on the function of the protein.

  If the useful protein is a grade that is useful for pharmaceutical use and has no problem for pharmaceutical use, it is desirable that the substance to be added is listed in the Japanese Pharmacopoeia or a pharmaceutical additive grade. However, it is not essential that all substances are listed in the Japanese Pharmacopoeia or pharmaceutical additive grades.

  In addition, the storage stability of enzymes used for various measurement and diagnosis purposes in addition to pharmaceutical use can be improved by using the storage stability improving method and composition disclosed by the present invention. It can be expected that it can be used for a long time.

  Useful protein compositions containing a compound having a thiol structure can contain other compounds that do not inhibit the activity of proteins, including other organic acids and organic acid alkali metal salts, and ingredients such as gum arabic. For example, polysaccharides other than gum arabic, polyols such as polyethylene glycol, saccharides such as sorbitol, amino acids such as glycine, or proteins such as gelatin may be added. Preferred are gum arabic, polyethylene glycol, and glycine.

  A surfactant may be added. The surfactant is preferably at least one selected from polyoxyethylene hydrogenated castor oil and polyoxyethylene sorbitan fatty acid ester.

  Moreover, since there is no problem even if sodium chloride is added, for example, when the composition is used as an injectable drug, the osmotic pressure can be adjusted with sodium chloride. In particular, in order to stably store a low concentration of useful protein in solution or as a lyophilized product, in addition to a compound having a thiol structure, any compound that does not inhibit the activity of the protein as described above can be included. Further, it is useful to add a surfactant. Also in these cases, by removing oxygen from the composition, it becomes possible to suppress the oxidation of the composition and to stabilize it.

  As described above, the useful protein composition of the present invention and the method for producing the useful protein composition are not only used for using the useful protein solution in a liquid state, but also after lyophilizing the useful protein solution into a preparation, It is also useful in applications where it is redissolved by, for example, injectable pharmaceuticals, diagnostics and the like. As a solvent for redissolving, water, buffer solution, physiological saline, or the like is preferably used.

  In the present invention, the production method of the useful protein is not limited. For example, the protein is produced by any method such as an organic synthesis method, a protein produced by using recombinant DNA technology, or a protein produced by extraction / purification from a natural product. Applicable to proteins. For example, it can be preferably applied to stabilization of canine interferon-γ and cat interferon ω produced by Escherichia coli or silkworm as useful proteins.

  Hereinafter, the present invention will be specifically described with reference to examples. The nitrogen gas used was high purity nitrogen (oxygen concentration of 10 ppm or less).

Reference example <Creation of canine interferon-γ by silkworm>
A virus solution of recombinant baculovirus rBNVγ disclosed in Examples 1 to 10 of JP-A-9-234085 is inoculated into silkworm larvae on the 5th and 2nd days, and commercially available artificial feed (Kanebo) at 25 ° C. for 4 days. The silk elegance company) was given and raised. 50 silkworm abdominals were cut and immersed in 500 ml of 50 mM acetate buffer (pH 3.5) containing 0.01% benzalkonium chloride and kept at 4 ° C. for 20 hours. The obtained silkworm body fluid extract was neutralized with 2N NaOH and then centrifuged at 5000 rpm for 15 minutes to recover the supernatant. The obtained supernatant was subjected to ultrafiltration using a holofiber type ultrafiltration membrane device (Amicon, molecular weight fraction size 100,000, HIP40-100). After passing this treatment solution through a copper chelate column, it is applied to a Q Sepharose column, washed with 20 mM glycine buffer (pH 9.0), and the adsorbate is eluted with a linear concentration gradient of sodium chloride. The fraction containing canine IFN-γ was recovered from the eluted fraction by reverse layer HPLC analysis, applied to a blue sepharose column, washed with 20 mM acetate buffer (pH 5.5), and then the linear concentration of sodium chloride. The adsorbate is eluted with a gradient. From the eluted fraction, a fraction containing canine IFN-γ was collected by reverse layer HPLC analysis, and this was desalted and used as a canine IFN-γ sample for examination of a stabilizer.

<Measurement method of antiviral activity>
The activity of canine interferon-γ was measured as the antiviral activity by the CPE method according to the literature of Prober et al, Science 238, 336-341 (1987). Vesicular Stomatitis Virus was used as a virus for measurement, and canine MDCK (ATCC CCL-34) cells were used as sensitive cells. That is, a diluted solution of a sample containing canine IFN-γ was added to canine MDCK cells cultured at 37 ° C. until confluent on a 96-well microplate, and further cultured at 37 ° C. for 20 to 24 hours to obtain antiviral activity. Was induced. VSV was further added, and the cells were cultured at 37 ° C. for 16 to 20 hours. Then, the surviving canine MDCK cells adhering to the microplate were stained with a crystal violet staining solution containing 20% formalin. The amount of crystal violet on the microplate was measured by measuring the absorbance at 570 nm, and the amount of canine IFN-γ when the cells were viable 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%.

<Method for measuring cysteine and cystine>
Cysteine and cystine were quantified by HPLC based on the literature of Junichi Koyama et al., Analytical Chemistry Vol. 37, 142-146 (1988). That is, an ultraviolet spectrophotometric detector (detection wavelength 210 nm) manufactured by Shimadzu Corporation was connected to a high-performance liquid chromatograph manufactured by Shimadzu Corporation. The separation column used was a CAPCELL PAK C18 manufactured by Shiseido with a particle size of 5 μm, and this was kept at 40 ° C. As the mobile phase, 0.1% (V / V) phosphoric acid, 5% (V / V) acetonitrile, and 3.5 mM aqueous sodium heptanesulfonate were used. The flow rate of the mobile phase was 0.4 mL / min.

  It is possible to quantify cysteine and cystine contained in a sample by analyzing a known concentration of cysteine and cystine aqueous solution in advance and drawing a calibration curve.

<Method for measuring dissolved oxygen concentration>
The dissolved oxygen in the liquid was measured with a portable multimeter manufactured by Thermo Fisher Scientific Co., Ltd.

Example 1 and Comparative Example 1
Gum arabic, cysteine, polyoxyethylene hydrogenated castor oil, trisodium citrate, citric acid, sodium acetate, polyethylene glycol, glycine were added to canine IFN-γ samples prepared according to <Creation of canine interferon-γ by silkworm> The final concentrations are 10 g / L, 0.48 g / L, 0.1 g / L, 3.79 g / L, 0.44 g / L, 0.21 g / L, 5 g / L, and 0.74 g / L, respectively. added. Two of these were prepared, and one of them was bubbled with nitrogen gas in a volume ratio of about 25 times the composition liquid over about 20 minutes. The other one did not bubble.
The results of measuring these dissolved oxygen concentrations are shown in Table 1.

  These were stored for 7 days at 4 ° C., and then the cysteine and cystine concentrations were measured. The results are shown in Table 2.

  From Table 1, a significant decrease in dissolved oxygen was observed by nitrogen bubbling. As a result, as shown in Table 2, the production of cystine could be remarkably suppressed.

Example 2 and Comparative Example 2
Two compositions were prepared in the same manner as shown in Example 1, and one of them was bubbled with nitrogen gas in a volume ratio of about 50 times the composition liquid over about 40 minutes. The other one did not bubble.

  The results of measuring these dissolved oxygen concentrations are shown in Table 3.

  These were stored for 24 days at 4 ° C., and then cysteine and cystine concentrations were measured. The results are shown in Table 4.

  Further, in order to confirm that bubbling nitrogen does not adversely affect the antiviral activity, antiviral activity was measured immediately after bubbling. The results are shown in Table 5.

  From Table 4, the production of cystine was remarkably suppressed by a large amount of nitrogen bubbling as compared with Example 1. There was a large amount of cystine precipitate in the sample that was not nitrogen bubbled. Furthermore, from Table 5, it was confirmed that nitrogen bubbling did not affect the antiviral activity.

Example 3 and Comparative Example 3
Two types of compositions (with / without nitrogen bubbling) shown in Example 2 were stored at 4 ° C. for 7 days after preparation, and then freeze-dried. Cysteine, cystine concentration and antiviral activity were measured on the lyophilized product after adding 1.2 times the volume of physiological saline prior to lyophilization to restore the aqueous solution. The results are shown in Table 6 and Table 7.

  From Table 6, the production of cystine was remarkably suppressed by a large amount of nitrogen bubbling even in the lyophilized preparation. Furthermore, it can be reconfirmed from Table 7 that nitrogen bubbling does not affect the antiviral activity.

Example 4 and Comparative Example 4
The lyophilized product reconstituted in aqueous solution shown in Example 3 was stored at 4 ° C., and the occurrence of precipitation and the time course of cysteine, cystine concentration and antiviral activity were measured. The results are shown in Table 8, FIG. 1 and FIG.

  As shown in Table 8, a significant change was observed on the sedimentation date of the stored material depending on the presence or absence of nitrogen bubbling. In the sample not subjected to nitrogen bubbling, precipitation began to occur in 7 days, whereas in the sample subjected to nitrogen bubbling, precipitation did not occur until 77 days had passed.

  In addition, it was clear that the generation of cystine was significantly suppressed by nitrogen bubbling and the stability was maintained at the cysteine and cystine concentrations shown in FIG. Further, in the antiviral activity, a clear decrease in activity was observed at 14 days in the sample not subjected to nitrogen bubbling, whereas the initial activity was maintained at 84 days in the sample subjected to nitrogen bubbling.

Claims (9)

A useful protein composition comprising canine interferon-γ, trisodium citrate or citric acid and cysteine, and a dissolved oxygen concentration of 3 mg / L or less. The useful protein composition according to claim 1, wherein the cysteine concentration is 0.1 to 0.8 g / L. The useful protein composition according to claim 1 or 2, wherein the concentration of cystine is 40% by mass or less of the total amount of cysteine and cystine present. The useful protein composition according to any one of claims 1 to 3, comprising at least one of a surfactant, polyethylene glycol, glycine, sodium chloride and gum arabic. The manufacturing method of the useful protein composition which freeze-drys the useful protein composition in any one of Claims 1-4. The method for producing a useful protein composition according to claim 5, wherein the solution is restored to a solution after lyophilization. An injectable pharmaceutical composition comprising the useful protein composition according to any one of claims 1 to 4 . A method for producing a useful protein composition , comprising contacting nitrogen gas with a composition containing canine interferon-γ, trisodium citrate or citric acid, and cysteine. The method for producing a useful protein composition according to claim 8, wherein nitrogen gas is bubbled throughout the useful protein composition.

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