KR20060061225A - Formulation of SEC1 Mutant Protein and Formulation Method thereof - Google Patents

Abstract

The present invention relates to the formulation of Staphylococcal enterotoxin C1 (SEC1) mutant protein, which is one of Staphylococcus aureus toxins, which has an excellent effect on the prevention, symptom relief and treatment of mastitis by improving the immune function of dry or non-organic cows. And solid phase microparticles containing SEC1 mutant protein, protein stabilizing excipient, carbohydrate auxiliary excipient and lipophilic substance as active ingredients are dispersed in biocompatible oil and / or fatty acid ester compound.

The formulation according to the present invention is effectively delivered to the body while maintaining the activity of the water-soluble mutant protein as an active ingredient, and has an excellent effect on the prevention and treatment of mastitis by increasing the immune effect when administered to cows due to the excellent antibody formation ability, It is also excellent in terms of its ability to be used as an injection.

Description

Formulation of SEC1 Mutated Protein and Method for Formulation of the Same

1 is a graph showing the results of measuring γ-IFN using a mouse cytokine ELISA kit from blood collected after injecting the formulations of the Examples and Comparative Examples according to the present invention;

Figure 2 is injected into the formulation of Example 1 according to the invention in non-organic cows having a somatic cell count of 500,000 / ㎖ or more and the milk of the cow is collected five times up to 2 weeks, 4 weeks, 6 weeks and 10 weeks before administration of the formulation Is a graph showing the results of measuring changes in somatic cell counts in milk;

Figure 3 is a formulation of Example 1 according to the present invention and the Laback staff (trade name) of Boehringer Ingelheim Co., Ltd. injected into non-organic cows having a somatic cell count of 500,000 / ㎖ or more to collect the cow's milk to obtain the somatic cell count in the milk It is a graph showing the result of the measurement.

The present invention relates to a formulation of Staphylococcal enterotoxin C1 (SEC1) mutant protein, which is one of the toxins of Staphylococcus aureus, which exhibits prevention, symptom relief and excellent therapeutic effect of mastitis through improved immune function of dry or non-organic cows and its A method for formulating, more specifically, an effective amount of a SEC1 variant protein; Protein stabilizing excipients; Carbohydrate-based auxiliary excipients; Lipophilic substances; And a biocompatible oil and / or a fatty acid ester compound, thereby providing a formulation of the SEC1 mutant protein which is easy to administer by injection and maximizes efficacy and stability, and a method of formulating the same.

The SEC1 variant protein, one of the toxins of Staphylococcus aureus, substitutes serine for cysteine, the 95th amino acid of Staphylococcus aureus C1, to produce antibodies specific to humoral immunity. However, the possibility of applying the effect as a vaccine for inducing the promotion of nonspecific cellular immunity is known (Terence N. Turner et al. (1992), Infection and immunity 62 (2); 694-697., Carolyn J. Hovde et al. (1994) Molecular Microbiology 13 (5); 897-909., Marcy L. Hoffann et al. (1994), Infection and Immunity 62 (8); 3396-3407). Methods for preparing SEC1 mutant proteins are disclosed in Korean Patent No. 382,239, Australian Patent No. 2001-11759, and the like.

As disclosed in Korean Patent No. 382,239, the SEC1 mutant protein having such mastitis prevention, symptom relief, and therapeutic effects can be mass-produced with E. coli as a host, but like other protein medicines for a long time (more than 2 weeks) There are problems in terms of maintaining the stability of the protein, such as degeneration of SEC1 mutant protein and aggregation of protein in a dispersion medium (oil, etc.) during storage. In addition, SEC1 mutant proteins, like common proteins, are unstable in heat, pH, salinity, organic solvents, and the like (Weiqi Lu et al. PDA L. Pharm. Sci. Tech. 49, 13-19 (1995)).

Meanwhile, the applicant's Korean Patent No. 359,252 discloses a method of preparing microparticles by spray drying using 3% carboxymethylcellulose and 2% lecithin to prepare microparticles of SEC1 mutant protein. . However, this method has some problems. The production of SEC1 mutant protein by spray drying is about 10-30%, which makes it unsuitable for commercialization by mass production. Since the temperature is 50 ~ 70 ℃ can affect the degeneration of the protein. For this reason, it is required to develop a formulation capable of effectively delivering in vivo after administration, while maintaining SEC1 mutant protein in a stable state for a sufficient time in an external environment, and to develop a technology capable of mass production.

For that reason, many studies have been conducted to formulate biodegradable polymers such as polyester-based polyglycolide, polylactide, and the like into solid phase microparticles. These formulations can maximize the effect by showing the effect of defense against degrading enzymes and the duration of drug effects in the body, but due to the property that the biodegradable polymer dissolves only in organic solvents during the formulation process, Since there is a problem that denaturation is caused, it is difficult to put it to practical use. Although another formulation for oral administration using liposomes has been attempted, it has not been put to practical use due to instability of particle structure and economical efficiency as an animal preparation.

The present invention aims to solve these problems of the prior art and technical problems that have been requested from the past.

After numerous experiments and in-depth studies, the present inventors have included an effective amount of SEC1 mutant protein as an active ingredient, and a solid-state microparticle by mixing a specific protein-stabilizing excipient, a carbohydrate-based excipient, and a lipophilic substance. Formulations prepared by dispersing them in biocompatible oils and / or fatty acid ester compounds (dispersion medium) are used for long-term storage of SEC1 mutant protein in solution, coagulation in dispersion medium, and various external conditions. Instability of the protein due to the field can be prevented, and it was confirmed that not only can maximize the prevention, symptom relief and therapeutic effect of mastitis in the body, but also confirmed that the commercialization by mass production is possible, and completed the present invention. It came to the following.

That is, the first object of the present invention is to provide a formulation capable of easily injecting SEC1 mutant protein, which is a water-soluble protein, while maintaining stability and long-term activity in vivo upon administration.

It is a second object of the present invention to provide a preparation method which enables commercialization of the formulation effectively through mass production.

Formulation according to the present invention for achieving the above object, 0.001 to 50% by weight of the SEC1 (Staphylococcal enterotoxin C1) variant protein, one of the toxins of Staphylococcus aureus as an active ingredient, 0.1 to 90% by weight protein stabilizing excipient, carbohydrate-based The solid phase microparticles containing 0.1 to 90% by weight of the auxiliary excipient and 0.1 to 10% by weight of the lipophilic substance are dispersed in the biocompatible oil and / or fatty acid ester compound.

The formulations according to the invention are particularly suitable for injection and exhibit high drug sustainability and good stability.

As described above, the SEC1 mutant protein, as described above, is an active ingredient exhibiting an excellent effect on the prevention, symptom relief and treatment of mastitis by improving the immune function of dry or non-organic cows, and those prepared by various methods may be used. have. The content of the active ingredient is 0.001 to 50% by weight based on the weight of the solid fine particles, as defined above, if too small content is difficult to exhibit a pharmacological effect, on the contrary, if too large, aggregation and denaturation in a water-insoluble solvent May occur. More preferred content is 0.01 to 20% by weight.

The formulations of the present invention contain specific ingredients for the purpose of enhancing the physicochemical stability of the water soluble mutant protein and producing stabilized solid phase microparticles.

Among such components, the protein stabilizing excipient is a component that can be granulated while stabilizing the active ingredient SEC1 variant protein.

To identify excipients that can prepare SEC1 mutant proteins into stabilized solid-phase microparticles, we selected excipient candidates that are likely to be predicted first in a number of excipients known to stabilize the protein, Confirmation experiments were performed on the formation of solid microparticles and protein stability. Microparticulation was performed by lyophilization in consideration of the problems of spray drying as described above.

 Table 1 below shows whether solid phase particles are formed when the mixture of SEC1 mutant protein and these excipients is lyophilized, and the formulation in which such solid fine particles are dispersed in oil is subjected to room temperature conditions (25 ° C., 60% RH) and harsh conditions ( Experimental results for the rate of change in protein purity when stored for 4 weeks at 40 ° C., 75% RH) are described. In addition, the results of dispersing only the water-soluble SEC1 variant protein in oil are also described for comparison.

As shown in Table 1, it can be seen that silica and calcium phosphate among the excipients used in the experiment do not form solid particles of the water-soluble SEC1 mutant protein. Among the excipients that form the solid particles, sodium chloride, polyethylene glycol 8000 shows the best stability to the protein, lactose, triple block conjugate (pluronic), polymethyl methacrylate, glucose, sugar, tetramethyl It can be seen that glucose and the like generally exhibit excellent stability against SEC1 mutant proteins.

From the above results, preferred protein stabilizing excipients that can be used in the formulation of the present invention include sodium chloride, polyethylene glycol (eg PEG 8000), disaccharides (eg lactose, maltose, sucrose, etc.), glucose One or two or more mixtures selected from the group consisting of, tetramethylglucose, and pluronic (triblock copolymer) may be preferably used, but is not limited thereto. Especially, polyethyleneglycol is more preferable and the mixture of polyethyleneglycol and sodium chloride is especially preferable.

The content of the protein stabilizing excipient is 0.1 to 90% by weight based on the weight of the solid fine particles as defined above, if too small content is difficult to exert the effect of the addition, on the contrary too much damage to the protein during the lyophilization process And after lyophilization may result in aggregation and denaturation of the particles in a non-aqueous solvent. More preferred content is 30 to 60% by weight.

The carbohydrate-based auxiliary excipient, which is another component used in the formulation of the present invention, assists the action of the protein stabilizing excipient and plays a role of maximizing the immune effect of the active ingredient SEC1 mutant protein, but is not limited thereto. , Preferably, one selected from the group consisting of sodium carboxymethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, chitosan, alzite, xylose, galactose, fructose, saccharose, dextran, and chondroitin cellulose Mixtures of two or more may be used. Especially, carboxymethylcellulose is especially preferable.

The content of the carbohydrate-based auxiliary excipient is 0.1 to 90% by weight based on the weight of the solid fine particles as defined above, if the content is too small it is difficult to exert the effect of the addition, if too large solid particles during lyophilization It may not be formed. More preferred content is 0.5 to 50% by weight.

Since the lipophilic substance is added to the formulation of the present invention, the lipophilic substance serves to improve the dispersibility of the microparticles containing the active ingredient SEC1 mutant protein and thus improve the injectability of the formulation. Preferably, but not limited to, phosphatidylserine, phosphatidylethanolamine, lecithin, phosphatidylcholine-based substances (eg, stearoyl phosphatidylcholine, arachidyl phosphatidylcholine), myristic acid, parmitic acid, stearic acid, sorbitan One or two or more mixtures selected from the group consisting of imitation oleate, polysorbate, glyceryl stearate, sorbitan palmitate, and sorbitan stearate can be used. Especially, the phosphatidylcholine type substance is especially preferable.

The content of the lipophilic substance is 0.1 to 10% by weight based on the weight of the solid fine particles, as defined above, if the content is too small it is difficult to exert the effect of the addition, on the contrary too much solid particles are formed after lyophilization It may not be. Preferred content is from 0.1 to 5% by weight.

The biocompatible oil is one of the media (dispersion media) capable of dispersing the solid phase microparticles containing SEC1 mutant protein, protein stabilizing excipient, carbohydrate auxiliary excipient and lipophilic substance as an injectable formulation. Although not limited, preferably one or two or more mixtures selected from the group consisting of cooking oil, minerals, oils, squalene, squalane and mono-, di- and triglycerides can be used. Examples of the cooking oil include soybean oil, jade oil, olive oil, safflower oil, cottonseed oil, peanut oil, sesame oil, sunflower oil and the like, and soybean oil is particularly preferable.

The fatty acid ester compound, which is another substance that can be used as the dispersion medium of the solid fine particles, is not limited to the following, but preferably monoglyceride, diglyceride, isopropyl palmitate, isopropyl myristate, triglyceride, One or two or more mixtures selected from the group consisting of benzoic acid benzoate, ethyl linoleate can be used. Especially, isopropyl myristate is preferable.

The biocompatible oils and fatty acid ester compounds may be used alone or in combination. The mixed use thereof as the dispersing medium is more preferable in terms of improving the injection performance and maximizing the dispersing effect, which can be confirmed from the results of Experimental Example 3 described later. In particular, in the case of using a mixture of soybean oil in the biocompatible oil and isopropyl myristate in the fatty acid ester compound, the injection performance of the formulation is more excellent.

For mixed use, for example, the biocompatible oil may be comprised between 1 and 99% by weight based on the total weight of the dispersion medium. When using such a mixture, when isopropyl myristate is used as a fatty acid ester compound, the content of especially preferable isopropyl myristate is 20 to 40 weight%.

The amount of the microparticles added to the dispersion medium is preferably 1 to 99% based on the total volume, in consideration of an appropriate single injection amount, the scanning ability of the dispersion, and the like. In some cases, a formulation in which the dispersion is dispersed again in saline or the like is also possible.

The formulation according to the present invention may be added with other ingredients known in the art without departing from the effect of the present invention, all of which should be construed as being included in the scope of the present invention.

The present invention also provides a method of preparing the SEC1 variant protein formulation. Method for producing a formulation according to the invention,

(a) mixing SEC1 variant protein, protein stabilizing excipient, carbohydrate-based auxiliary excipient, and lipophilic material;

(b) lyophilizing the mixture obtained above to prepare solid fine particles; And

(c) dispersing the solid phase microparticles in a biocompatible oil and / or a fatty acid ester compound;

It is configured to include.

In the step (b), the solid fine particles are produced to have a particle diameter of approximately 5 to 200 ㎛. If the particle size is too small, it is difficult to achieve dispersion and slow release of the active ingredient, and on the contrary, if the particle size is too large, particles are precipitated in the dispersion medium, which is not preferable because it is difficult to maintain the dispersed state.

Lyophilization method for the production of the microparticles is known in the art, detailed description thereof is omitted herein.

 Hereinafter, although this invention is demonstrated in detail with reference to an Example, since these are only illustrations of this invention, the scope of the present invention is not limited by it.

Examples and Comparative Examples: Preparation of SEC1 Mutant Protein Formulations

SEC1 variant protein formulations were prepared with the composition as shown in Table 2 below. For example, in Example 1, SEC1 mutant protein, sodium chloride, carboxymethylcellulose and phosphatidylcholine are mixed and then lyophilized to prepare solid microparticles having an average particle diameter of about 50 to 80 μm, which are dispersed in soybean oil. Formulations were made.

Experimental Example 1: Stability test of formulation containing SEC1 mutant protein for injection

When the formulations of Examples 1-3 and Comparative Examples 1-3 were used as injectable formulations, mice were tested to determine whether the SEC1 variant protein in the formulation had activity in the body. Specifically, the formulation was added to soybean oil, diluted to a concentration of 40 μg of SEC1 mutant protein, and intraperitoneally injected into four-week-old male Balb / c mice, respectively, at 0, 2, 4, 8, 16, and 24 days later. Was taken. Γ-IFN was measured from the blood using a mouse cytokine ELISA kit. The measured results are shown in FIG. 1.

As shown in FIG. 1, γ-IFN levels were significantly higher than those of 40 μg of the original agent in the order of Example 1, Example 3, and Example 2 up to 24 days after administration of the formulation, and showed excellent sustainability. On the contrary, in Comparative Example 1, the γ-IFN level was increased up to 8 days after administration, but decreased after 8 days. Comparing the results of Example 1 and Comparative Example 1, it can be seen that carboxymethylcellulose, a carbohydrate-based excipient, maximizes the immune effect of SEC1 mutant protein in vivo. In addition, when comparing the results of Example 1 with the results of Comparative Example 2 and Comparative Example 3, the formulations of Comparative Examples 2 and 3 increased rapidly up to 8 days after administration, which was sharply reduced, which is the present invention in terms of stability of the SEC1 variant protein. Means that the compositions are essential.

Experimental Example 2: Injectability Test of Formulations Containing Injectable SEC1 Mutant Protein

In order to quantitatively evaluate whether the solid-state microparticles containing the SEC1 mutant protein of the present invention are uniformly dispersed in a non-aqueous solution or a mixture of a non-aqueous solution and an aqueous solution, the respective formulations (dispersions) may be injected by syringe. The force at the time (scanning power) was measured. Specifically, 0 and 28 days after storage of the force (scanning ability) required for extrusion when the syringe (injection needle: 18 gauge) filled with 3 ml of each dispersion was pushed at a constant speed of 80 mm / min. Were measured respectively. For comparison, soybean oil was used as a control, and a formulation in which only phosphatidylcholine was excluded from the components of Example 1 was prepared and used as Comparative Example 4. The scanning ability of these dispersions is shown in Table 3 below.

As shown in Table 3, among the dispersions 28 days after storage of the formulation, the dispersions of Examples 1, 2 and 3 and Comparative Example 1 were excellent in dispersibility and were easy to inject like soybean oil as a control. On the other hand, it can be seen that the dispersions of Comparative Examples 2 and 3 have difficulty in injection or are not injected. In particular, it can be seen that the dispersion (Comparative Example 4) containing no lipophilic substance in the present invention is not injected.

Experimental Example  3: for injection SEC1  Of formulations containing variant proteins Injection  Experiment

Test conditions were the same as Experimental Example 2 and measured after 0 and 24 weeks after storage, respectively. For comparison, the injectability of these dispersions is shown in Table 4 below using Example 1, Examples 4, 5, and 6 in which soybean oil and isopropyl myristate are mixed.

As shown in Table 4, the dispersion of Examples 4, 5, 6 of the dispersion at 24 weeks after the formulation storage can be seen that the effect of significantly improving the injection performance and dispersibility of Example 1. Based on the above results, it can be seen that when the biocompatible oil (especially soybean oil) is mixed with the fatty acid ester compound, the injection performance is further improved. In particular, in the case of isopropyl myristate, it may be more preferable when used in an amount of 20 to 40% by weight based on the total weight of the dispersion medium.

Experimental Example  4: for injection depending on the concentration of sodium chloride SEC1  Stability Test of Mutant Proteins

Table 5 below shows the formulations in which solid phase particles were dispersed in oil when the mixture of SEC1 mutant protein and these excipients were lyophilized for 24 weeks at room temperature (25 ° C., 60% RH) and harsh conditions (40 ° C., 75% RH). The protein content when stored for a while is described. In addition, in order to compare the change in protein content according to the content of sodium chloride, the protein content results of Examples 7 to 10 are described together.

As shown in Table 5, the formulation of Example 1 showed a stable result without changing the content for 24 weeks at room temperature and harsh conditions, the formulations of Examples 7 to 10 showed a tendency to decrease the protein content . These results show that the sodium chloride content of solid phase particles of SEC1 mutant protein affects the stability of the protein. Thus, when sodium chloride is used as the protein stabilizing excipient, it can be seen that its particularly preferred content is 60% by weight or less. However, the experimental results show that the formulations of the present invention, including the formulations of Examples 7-10, have excellent overall stability of SEC1 variant proteins even when stored for extended periods of time (24 weeks) in harsh conditions (40 ° C., 75% RH). To ensure that it is guaranteed.

Experimental Example 5: Antibody-producing ability of the formulation containing the SEC1 mutant protein for injection

In order to investigate the biological activity of animal intraperitoneal administration of the formulation of Example 1 based on the results of Experiment 1, 0.3 ml (400 µg) of the formulation of Example 1 was diluted 10-fold in soybean oil and 0.3 ml (40 µg). And 0.03 ml (4 μg) prepared at 100-fold dilution were immunized three times at two week intervals in the abdominal cavity of mice. Blood was collected from mice (10 mice / tea) after 14 days for every 1st, 2nd and 3rd doses and serum was isolated, using a peroxidase (ICN. # 55550) conjugated with goat-derived anti mouse IgG, SEC1 variant protein. The titers of antibodies specific for were analyzed. The results are shown in Table 6 below.

As shown in Table 6, it can be seen that the formulation of Example 1 shows an excellent antibody production capacity with respect to the content of SEC1 mutant protein in vivo in the mouse.

Experimental Example 6: Experiment of somatic cell number reduction effect of the formulation containing SEC1 mutant protein for injection

As a somatic cell experiment of a target animal for verifying the immune enhancing effect of cows, the formulation of Example 1 was intramuscularly injected into 295 non-organic cows having a somatic cell count of 500,000 / ml, and 2 weeks after administration of the cow's milk. 5 times, 4 weeks, 6 weeks, and 10 weeks were collected to determine the change in somatic cell count in milk. The results are shown in FIG.

As shown in Figure 2, it can be seen that the formulation of Example 1 shows an effect of reducing somatic cell count in milk continuously from 4 weeks to 10 weeks after administration.

Experimental Example 7: Comparison of somatic cell count reduction effect of the formulation containing the SEC1 mutant protein for injection

The somatic cell experiment of the subject animal was carried out by comparing the immune enhancing effect of the cow of the formulation of Example 1 with Laback Staff (trade name), the cow's Staphylococcus aureus mastitis vaccine from Boehringer Ingelheim, as a comparative example. 295 non-organic cows having a concentration of / ml or more were injected intramuscularly into 278 cows of the formulation of Example 1 and 17 cows of the formulation of the comparative example, and the milk of the cows was collected to measure the somatic cell count in the milk. The results are shown in FIG.

As shown in Figure 3, the formulation of Example 1 showed better results in the rate of somatic cell loss in milk compared to Labag staff of the comparative example.

Experimental Example 8: Comparative experiment of SEC1 mutant protein microparticle production by lyophilization and spray drying

 Microparticles of SEC1 mutant protein were prepared by the freeze-drying method having the most ideal eutectic point condition in which the stability of SEC1 mutant protein was maintained and the spray drying method described in Korean Patent No. 359,252. It was. Experimental conditions and their results are set forth in Table 5 below.

As shown in Table 7, the production rate by the spray drying method is about 11%, whereas the production rate (yield%) by the freeze-drying method according to the present invention can be seen that a very big difference as about 99%. . That is, in the case of SEC1 mutant protein, it can be seen that only the lyophilization method is suitable for commercialization for mass production.

As described above, the SEC1 mutant protein-containing formulation according to the present invention includes a protein stabilizing excipient, a carbohydrate auxiliary excipient, a lipophilic substance and a dispersion medium to effectively deliver the soluble mutant protein to the body while maintaining the activity of the soluble mutant protein, and with excellent antibody formation ability. Due to the increased immune effect when administered to the cow has an excellent effect on the prevention and treatment of mastitis, it can be used as an injection because it is excellent in terms of injection capacity.

Those skilled in the art to which the present invention pertains will be able to make various applications and modifications within the scope of the present invention based on the above contents.

Claims (16)

0.001-50% by weight of Staphylococcal enterotoxin C1 (SEC1) variant protein, one of Staphylococcus aureus toxins, 0.1-90% by weight protein stabilizing excipient, 0.1-90% by weight carbohydrate auxiliary excipient and 0.1-90% lipophilic substance SEC1 mutant protein formulation comprising 10% by weight and consisting of lyophilized solid phase microparticles dispersed in a biocompatible oil and / or fatty acid ester compound. The method of claim 1, wherein the protein stabilizing excipient, sodium chloride, polyethylene glycol, disaccharides, glucose, tetramethyl glucose, and Pluronic (triblock copolymer), characterized in that one or two or more mixtures selected from the group consisting of SEC1 variant protein formulation. The carbohydrate-based excipient is sodium carboxymethylcellulose, carboxymethylcellulose, hydrophyllulose, chitosan, alzite, xylose, galactose, fructose, saccharose, dextran, and chondroitin cellulose. SEC1 variant protein formulation, characterized in that one or more mixtures selected from the group consisting of. According to claim 1, wherein the lipophilic substance is phosphatidylserine, phosphatidylethanolamine, lecithin, phosphatidylcholine-based substance, myristic acid, palmitic acid, stearic acid, sorbitan monooleate, polysorbate SEC1 variant protein formulation, characterized in that one or more mixtures selected from the group consisting of, glyceryl stearate, sorbitan palmitate, and sorbitan stearate. According to claim 1, wherein the biocompatible oil is one or two selected from the group consisting of soybean oil, cottonseed oil, olive oil, sesame oil, corn oil, peanut oil, palm oil, minerals, squalene, squalane and mono-, di- and triglycerides SEC1 variant protein formulation, characterized in that the mixture of the above. The method of claim 1, wherein the fatty acid ester compound is one or two or more mixtures selected from the group consisting of monoglycerides, diglycerides, triglycerides, isopropyl palmitate, isopropyl myristate, benzoic acid benzene and ethyl linoleate SEC1 variant protein formulation. 2. The SEC1 variant protein formulation of claim 1, wherein the solid phase microparticles are dispersed in a mixture of biocompatible oils and fatty acid ester compounds. 3. The SEC1 variant protein formulation of claim 2, wherein the protein stabilizing excipient is sodium chloride and / or polyethylene glycol. 4. The SEC1 variant protein formulation of claim 3, wherein the carbohydrate-based excipient is carboxymethylcellulose. 5. The SEC1 variant protein formulation of claim 4, wherein the lipophilic material is a phosphatidylcholine-based material of 0.1 to 10% by weight. 9. The SEC1 variant protein formulation according to claim 8, wherein the content of the protein stabilizing excipient sodium chloride and / or polyethylene glycol is 30 to 60% by weight based on the weight of the solid microparticles. 6. The SEC1 variant protein formulation of claim 5, wherein the biocompatible oil is soybean oil. 8. The SEC1 mutant protein formulation of claim 7, wherein the fatty acid ester compound is isopropyl myristate. According to claim 1, wherein the solid phase microparticles SEC1 variant protein formulation, characterized in that the average particle size of 5 to 200 ㎛. 14. The SEC1 variant protein formulation of claim 13, wherein the content of isopropyl myristate is 20 to 40% by weight based on the total weight of the dispersion medium. A method of preparing a SEC1 variant protein formulation according to claim 1 comprising the following steps: (a) mixing SEC1 variant protein, protein stabilizing excipient, carbohydrate-based auxiliary excipient, and lipophilic material; (b) lyophilizing the mixture obtained above to prepare solid fine particles; And (c) dispersing the solid microparticles in a biocompatible oil and / or a fatty acid ester compound.

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