KR102146555B1 - Pharmaceutical composition having anthrax-specific antimicrobial activity and method for producing the same - Google Patents

Abstract

Mixing anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution; Freezing the mixed mixture; First drying the frozen mixture; And repeatedly drying the first dried mixture two or more times, and the step of repeatedly drying the two or more times is a step of repeating drying while increasing a drying temperature, having an anthrax-specific antibacterial activity It provides a method for preparing a pharmaceutical composition, a pharmaceutical composition having an anthrax-specific antibacterial activity, and a drug comprising the same.

Description

Pharmaceutical composition having anthrax-specific antimicrobial activity and method for producing the same {Pharmaceutical composition having anthrax-specific antimicrobial activity and method for producing the same}

Mixing anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution, freezing the mixed mixture, first drying the frozen mixture, and twice or more of the first dried mixture Including the step of repeatedly drying, the step of repeatedly drying the two or more times, the step of repeating drying while increasing the drying temperature, a method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity, a pharmaceutical composition, and the above Disclosed is a drug having an anthrax-specific antimicrobial activity comprising the composition.

Anthracis (Bacillus anthracis) is a Gram-positive aerobic bacteria that forms spores and has no flagella and no mobility. In the blood and tissues of infected animals, it is observed as mono-, bi- or streptococcus, and does not form spores in vivo. Anthrax is the causative agent of an infectious disease known as'Anthrax' or'Anthrax'. Anthrax is an acute and febrile contagious disease that occurs mainly in herbivores and omnivores, and is a second-class legal infectious disease of livestock that causes sudden death due to fever and shortness of breath. Anthrax, which can infect humans, can be classified as a common infectious bacteria and can also be used as a biological weapon. Animal anthrax infections are mostly oral infections with soil or grass contaminated with anthrax, and skin infections or respiratory infections through wounds are common. In addition, bloodsucking insects can also act as a source of infection.

Anthrax can occur in most mammals, such as cattle, sheep, goats, mules, and dogs, and can also occur in humans if livestock products from infected animals are consumed. The incubation period of anthrax is 1 to 5 days, and clinical symptoms include deep acute plastic surgery, which dies 1 to 2 hours after the onset of symptoms, and acute type, which dies within 24 hours. In general, sudden fever, difficulty breathing, and depression can be seen.

The outbreak of anthrax in Korea has been common in animals from the first official report in 1907 to the 1930s, but in modern times, the incidence of anthrax in animals has rapidly decreased due to the development of vaccines, and few cases have recently been reported. Without.

However, since anthrax is highly likely to be abused as a biological weapon, the development of treatment technology related to anthrax needs to be continued. In biological terrorism, spores in the aerosol state are mainly used.In the case of terrorism using anthrax, anthrax toxin that enters the upper airway remains latent in the upper airway and then penetrates the lymphatic system around the lungs. It can cause swelling, etc., causing symptoms similar to colds in the beginning, eventually leading to death due to difficulty breathing. In addition, the Centers for Disease Control and Prevention (CDC) treats anthrax as the highest-risk bacterial species by including it in Category A pathogens.

For this reason, there is a need to prepare for biological terrorism or biological warfare caused by anthrax. However, since anthrax is evolving at a very rapid rate, it is not easy to prepare effective infection treatment measures. Antibiotic treatment, such as a conventional penicillin formulation, has a limited effect, and in the case of acute course, there is a problem that the therapeutic effect can hardly be expected.

Therefore, it is urgent to prepare a method that can provide a rapid therapeutic effect, and the development of a pharmaceutical formulation that can be effectively utilized is urgently needed. As a means of providing a rapid therapeutic effect, the present inventors have proposed the use of an antibacterial protein B4 that can provide a rapid lytic effect against anthrax bacteria. The antimicrobial protein B4 has excellent antibacterial activity against anthrax (Korean Patent Registration 10-1765393).

However, for the convenient and stable handling, transportation, storage, and use of antibacterial protein B4 in the medical field or in the battlefield, it is necessary to secure a freeze-dried formulation that is allowed to be handled at room temperature rather than a liquid frozen form or liquid refrigerated form. Do. It is common that a lyophilized formulation having a stable storage capacity is not achieved by applying the known composition and method as it was used to develop a lyophilized formulation of protein. Moreover, in the case of antibacterial protein B4, unlike conventional protein preparations such as antibody proteins or hormone preparations, the enzymatic activity must be well preserved, and structurally, it is somewhat complicated, such as having an active region and a cell wall binding region. In addition, unlike conventional protein preparations such as antibody proteins or hormone preparations, it has not been long since related research has been conducted, and since relevant information has not been sufficiently secured, considerable research and efforts are also required in developing freeze-dried formulations. Therefore, there is a need for technology development related to a pharmaceutical composition that can be easily stored in a lyophilized formulation and easily used in the field and a method of manufacturing the same.

Korean Patent Registration No.1765393, "Method for producing anthrax-specific lytic protein B4 and a method for treating diseases caused by anthrax" (published date: 2017.05.15)

It is an object of the present invention to mix the anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution, freezing the mixed mixture, first drying the frozen mixture, and the first drying Including the step of drying the mixture by repeating two or more times, and the step of repeatedly drying the mixture two or more times is a step of repeating drying while increasing a drying temperature, a method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity. To provide.

Another object of the present invention includes anthrax-specific antibacterial protein B4 and a stabilizing solution represented by SEQ ID NO: 1, and the stabilizing solution is histidine (L-Histidine) having a concentration of 1 mM to 50 mM, and a concentration of 0.05% (w/ v) to 10% (w / v) polysorbate 80 (polysorbate 80) and a concentration of 1% (w / v) to 10% (w / v) containing trehalose (trehalose), anthrax specific It is to provide a pharmaceutical composition having an antibacterial activity.

Another object of the present invention is to provide a drug having an anthrax-specific antibacterial activity comprising the composition.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

According to an embodiment of the present invention, mixing the anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution; Freezing the mixed mixture; First drying the frozen mixture; And repeatedly drying the first dried mixture two or more times, and the step of repeatedly drying the two or more times is a step of repeating drying while increasing a drying temperature, having an anthrax-specific antibacterial activity A method of preparing a pharmaceutical composition is provided.

According to one side, the stabilizing solution may include histidine (L-Histidine), polysorbate 80 (polysorbate 80), and trehalose.

According to one side, the stabilizing solution may be a solution of pH 5.0 to pH 9.0.

According to one side, the freezing may be performed in a temperature range of -90°C to -70°C.

According to one side, the primary drying may be performed in a temperature range of -30°C to -10°C.

According to one side, the manufacturing method may further include the step of obtaining an anthrax antibacterial protein B4 from Escherichia coli BL21-pBAD-B4 strain [accession number: KCTC 12709BP].

According to another embodiment of the present invention, anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1; And stabilizing solution; Including, the stabilization solution, histidine (L-Histidine) of a concentration of 1mM to 50mM; Polysorbate80 with a concentration of 0.05% (w/v) to 10% (w/v) and trehalose with a concentration of 1% (w/v) to 10% (w/v) ), comprising an anthrax-specific antibacterial activity is provided.

According to one side, the stabilizing solution may be a pH of 5.0 to pH 9.0.

According to one side, the anthrax-specific antibacterial protein B4 may be obtained from the Escherichia coli BL21-pBAD-B4 strain [accession number: KCTC 12709BP].

According to another embodiment of the present invention, there is provided a drug having an anthrax-specific antimicrobial activity comprising any one of the aforementioned pharmaceutical compositions having an anthrax-specific antibacterial activity.

By using the manufacturing method of the present invention, a pharmaceutical composition having an anthrax-specific antibacterial activity that can be stably stored in a lyophilized formulation can be prepared. The composition of the present invention and the formulation comprising the same provide high stability and convenience in handling, transportation, storage, and use. In particular, a frozen formulation that allows for room temperature handling unlike other liquid forms or general frozen formulations that require refrigeration while enhancing the preservation of a composition containing a protein that has properties that require good enzymatic activity, such as antibacterial protein B4. As it can be manufactured, it has the advantage of being easy to use in medical or battlefields.

However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

FIG. 1 shows the results of preparing an anthrax-specific antibacterial protein B4 used in the present invention as a recombinant protein by electrophoresis.
Figure 2 shows the results of analyzing the storage stability of the composition of the present invention compared to the control.
3 shows the results of comparing the biological activity of the composition of the present invention with a control group.

Hereinafter, exemplary embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the rights of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes to the embodiments are included in the scope of the rights.

The terms used in the examples are used for illustrative purposes only and should not be interpreted as limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

In addition, in the description with reference to the accompanying drawings, the same reference numerals are assigned to the same components regardless of the reference numerals, and redundant descriptions thereof will be omitted. In describing the embodiments, when it is determined that a detailed description of related known technologies may unnecessarily obscure the subject matter of the embodiments, the detailed description thereof will be omitted.

According to an embodiment of the present invention, mixing the anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution; Freezing the mixed mixture; First drying the frozen mixture; And repeatedly drying the first dried mixture two or more times, and the step of repeatedly drying the two or more times is a step of repeating drying while increasing a drying temperature, having an anthrax-specific antibacterial activity A method of preparing a pharmaceutical composition is provided.

According to another embodiment of the present invention, anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1; And stabilizing solution; Including, the stabilization solution, histidine (L-Histidine) of a concentration of 1mM to 50mM; Polysorbate80 with a concentration of 0.05% (w/v) to 10% (w/v) and trehalose with a concentration of 1% (w/v) to 10% (w/v) ), comprising an anthrax-specific antibacterial activity is provided.

The antimicrobial protein B4 is a protein having an activity capable of specifically lyzing anthrax bacteria and has an amino acid sequence represented by SEQ ID NO: 1, and the anthrax-specific antibacterial protein B4 is composed of 262 amino acids, and a molecular weight of about 27.9 kDa. Reaches.

In one case, the antimicrobial protein B4 may be an antibacterial protein B4 variant or an antibacterial protein B4 analog having predetermined mutations such as partial substitution, partial addition, and partial deletion of the amino acid sequence represented by SEQ ID NO: 1. The antimicrobial protein B4 may be composed of an amino acid sequence having 80% or more homology to the amino acid sequence represented by SEQ ID NO: 1. Specifically, the antimicrobial protein B4 may consist of an amino acid sequence having 85% or more homology to the amino acid sequence represented by SEQ ID NO: 1, and more specifically, the antibacterial protein B4 is an amino acid sequence represented by SEQ ID NO: 1 and It may consist of an amino acid sequence having 90% or more homology. Preferably, the antimicrobial protein B4 may consist of an amino acid sequence having 95% or more homology to the amino acid sequence represented by SEQ ID NO: 1, but the antibacterial protein B4 is most preferably composed of an amino acid sequence represented by SEQ ID NO: 1. Do.

The anthrax-specific antimicrobial protein B4 represented by SEQ ID NO: 1 was developed and produced by the present inventors and deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center as of November 12, 2014 (Escherichia coli BL21-pBAD-B4 , Accession number: KCTC 12709BP).

Using the above-described anthrax-specific antibacterial protein B4 and a stabilizing solution, a pharmaceutical composition having an anthrax-specific antibacterial activity can be prepared in the following process.

First, an anthrax-specific antibacterial protein B4 and a stabilizing solution are prepared. If necessary, the process of obtaining anthrax antibacterial protein B4 from the E. coli strain may be preceded.

The stabilizing solution is prepared to contain histidine (L-Histidine), polysorbate 80 (polysorbate80) and trehalose as active ingredients, and each of the active ingredients is prepared to have the following concentration range. desirable.

The histidine may have a concentration of 1mM to 50mM, specifically 1mM to 30Mm, more specifically 5mM to 15mM, but most preferably 10mM.

The polysorbate 80 may have a concentration of 0.05% (w/v) to 10% (w/v), and specifically 0.05% (w/v) to 5% (w/v). And, more specifically, it may be from 0.05% (w/v) to 1% (w/v), but it is most preferably 0.1% (w/v).

The trehalose may have a concentration of 1% (w/v) to 10% (w/v), and specifically, 3% (w/v) to 7% (w/v). Although preferred, 5% (w/v) is most preferred.

The acidity of the stabilizing solution may range from pH 5.0 to pH 9.0, which is based on a value measured before lyophilization. Specifically, the acidity of the stabilizing solution is preferably a solution of pH 5.0 to pH 7.0, but most preferably pH 6.0.

The antibacterial protein B4 prepared as above and the stabilizing solution are mixed to prepare a mixture solution. In this case, the concentration of the antimicrobial protein B4 to be mixed may be adjusted within a predetermined concentration range according to the purpose of the pharmaceutical composition. The predetermined concentration range may be 0.1mg/ml to 30mg/ml. Specifically, the concentration of the antimicrobial protein B4 is preferably 1mg/ml to 20mg/ml.

The mixture solution mixed as above is first stored and frozen for about one day. Thereafter, it is put in a freeze dryer to perform primary drying, and then drying is performed two or more times while increasing the temperature with a predetermined gap.

The freezing temperature may be -90°C to -70°C, specifically, -85°C to -75°C, but it is preferably -80°C. The freezing time may vary depending on the type and concentration of the components included in the mixture solution and the amount of the total solution, but it is preferably stored and frozen for 20 to 28 hours.

The primary drying temperature may be -30°C to -10°C, specifically, -25°C to -15°C, but is preferably -20°C. In this case, the drying time may also vary depending on the type and concentration of the components included in the mixture solution and the amount of the total solution, but drying is preferably performed for 12 to 20 hours.

The pharmaceutical composition having an anthrax-specific antimicrobial activity of the present invention prepared as described above may be used in a lyophilized form and re-dissolved using a pharmaceutically acceptable medium.

In this case, the term "pharmaceutically acceptable medium" means a carrier or diluent that does not impair the biological activity and properties of the administered drug, and is usually used in medical fields, such as water for injection or It may be normal saline.

The appropriate application amount, spray amount, or dosage of the solution prepared by re-dissolving may be factors such as formulation method, administration method, age, weight, severity of symptoms, food intake, administration time, administration route, excretion rate, and response sensitivity. They may be different from each other, and depending on the object, purpose or use, a drug containing an effective amount of the composition of the present invention described above can be prepared and used.

The pharmaceutical composition having an anthrax-specific antimicrobial activity of the present invention or a drug comprising the same specifically lyses anthrax, and thus can exhibit a therapeutic effect in the treatment of diseases caused by anthrax.

At this time, the disease caused by the anthrax is generically referred to as symptoms accompanied by fever and shortness of breath due to an anthrax infection. In addition, the treatment or treatment is meant to include all actions in the usual category of suppressing anthrax-induced disease and alleviating the pathological condition of anthrax-induced disease, and is not particularly limited.

The pharmaceutical composition having an anthrax-specific antibacterial activity or a drug containing the same may be used as, for example, antibiotics, disinfectants, disinfectants, therapeutic agents, etc., and may also be used to treat anthrax infection caused by war using biological terrorism or biochemical weapons. , The use is not limited thereto.

When using the pharmaceutical composition having an anthrax-specific antibacterial activity or a drug containing the same for treatment, a solution re-dissolved using a medium as described above is administered orally, parenterally, or nasal spray. The method can be used, and it can also be used by spraying into the environment.

A more detailed description will be described later through the following examples.

Example 1. Obtaining antibacterial protein B4

In order to obtain an antibacterial protein B4 having an anthrax-specific antimicrobial activity, an Escherichia coli strain (Escherichia coli BL21-pBAD-B4, accession no. 12709BP) was used as a production strain.

First, the Escherichia coli strain (Escherichia coli BL21-pBAD-B4) deposited in 20 ml of LB medium containing 50 μg/ml of kanamycin (trypton 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) 20 μl was added and inoculated, followed by shaking culture at 37° C. for about 6 to 7 hours. Thereafter, 20 μl of the cultured culture was inoculated into 200 ml of an LB medium containing 50 μg/ml kanamycin, followed by shaking culture at 37° C. for about 7 to 8 hours. Put the cultured culture medium into an incubator containing 5 L of LB medium containing 50 μg/ml kanamycin, add so that the OD ₆₀₀ value (absorbance at 600 nm) becomes 0.1, and then at a speed of 200 rpm, 5 L per minute Aeration of, and incubated at 37°C. When the OD ₆₀₀ value reached 0.5, L-arabinose was added so that the final concentration became 0.2% to induce the expression of anthrax-specific lytic protein B4 of the amino acid sequence represented by SEQ ID NO: 1 at 37°C. Incubation was further performed for 4 hours at.

After completion of the culture, the cell culture solution was taken and centrifuged at 6,000 rpm for 10 minutes at 4°C to recover the cell precipitate, and the recovered cell precipitate was suspended in 200 ml of 20 mM Tris-HCl (pH 8.0) buffer. After that, the suspended suspension was disrupted by ultrasonic grinding. In the ultrasonic pulverization method, the cells were broken by applying ultrasonic waves for 3 seconds, and the condition of stopping for 3 seconds was repeated for a total of 15 minutes, but was performed in an ice bath to prevent cell degeneration. The obtained cell lysate was centrifuged at 13,000 rpm for 20 minutes at 4° C., and the supernatant was purified. The purification process uses a conventional cation-exchange chromatography method, and the material used in the purification process and a simple implementation process are as follows.

As a cation-exchange resin, 5 ml of HiTrapTM SP HP (GE Healthcare) was used. Chromatography was carried out after pre-equilibrating the column with buffer A (20 mM Tris-HCl, pH 8.0), and the sample was dropped onto the column, and the flow rate of 5 ml/min. Buffer A was washed while flowing 5 CV (column volume). After washing, the concentration gradient from buffer A to buffer B (20 mM Tris-HCl, 500 mM sodium chloride, pH 8.0) at a flow rate of 5 ml/min is from 0% to 100%. Chromatography was performed under the conditions of becoming. In this process, elution of the anthrax-specific lytic protein B4 having the desired amino acid sequence represented by SEQ ID NO: 1 was achieved, and through this process, anthrax-specific lytic protein B4 could be obtained with a purity of 90% or more.

1 is a confirmation of the anthrax-specific antibacterial protein B4 prepared as described above, and the antibacterial protein B4 purified using a chromatography purification process was confirmed by electrophoresis. Lane M of FIG. 1 represents a protein size marker, and the size of the protein of the present invention loaded in column 2 was confirmed based on the protein size markers in column 1.

Example 2. Preparation of pharmaceutical composition having specific antibacterial activity for anthrax bacteria

A pharmaceutical composition of a freeze-dried formulation containing the antibacterial protein B4 prepared in Example 1 as an active ingredient, and improved handling and storage stability was prepared.

Various buffer conditions applicable to the pharmaceutical composition, various types of stabilizers, and various types of additives were applied to derive composition candidate samples of various compositions, and a solution sample was prepared by adding anthrax-specific antibacterial protein B4 to each sample. . Thereafter, it was stored at -80°C for one day and then frozen, put it in a freeze dryer, dried for 16 hours at -20°C, and dried repeatedly while increasing the temperature by 10°C every 1 hour. The temperature was kept at 20° C. for a total of 4 hours.

A method for preparing a composition suitable for antimicrobial protein B4 was developed by investigating the degree of stability maintenance of the antimicrobial protein B4 when the lyophilized product (lyophilized product) of the lyophilized candidate sample was stored at 37°C for 12 weeks as described above.

In particular, with regard to maintaining the stability of the antimicrobial protein B4, structural integrity is maintained at 90% or more, and the TOD ₅₀ value (the time it takes to decrease to half of the initial bacterial absorbance) as a biological activity is 20 of the initial value from the initial value. A method for preparing a composition was developed by setting the minimum target performance not to increase above %.

Specifically, a candidate sample solution containing antibacterial protein B4 prepared in various compositions was subjected to lyophilization in various ways (applying various conditions under detailed conditions such as drying step, application time of each step, temperature, etc.), and then obtained through When the lyophilisate was stored at 37°C for 12 weeks, the formulation was excellent in maintaining stability and the lyophilization method applied thereto was selected.

To investigate the structural stability of the antibacterial protein B4 in the lyophilized composition, size-exclusion high-performance liquid chromatography (size-exclusion HPLC) was used, and the biological activity stability of the antibacterial protein B4 in the lyophilized composition. Was investigated through a turbidity reduction assay. Here, structural stability refers to the degree to which no change in the antibacterial protein B4 is degraded, the antimicrobial protein B4 is aggregated, or the purity of the antimicrobial protein B4 is changed.

The structural stability investigation of the antibacterial protein B4 using size exclusion high performance liquid chromatography was carried out as follows. The column was a SEC column (Phenomenex column, #00H-2145-K0), and a mobile phase buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.0) was applied. During HPLC analysis, the sample loading volume was 50 μl, the flow rate was 1 ml/min, and the analysis time was a total of 30 minutes.

Example 3. Stability and biological activity verification of antibacterial protein B4

The biological activity of antimicrobial protein B4 using the turbidity reduction assay was investigated as follows.

In this experiment, Bacillus cereus, an anthrax-like strain, was used. This alternative use of Bacillus Sirius, an anthrax-like strain, was taken because of the convenience of the experiment because the antibacterial activity of the antibacterial protein B4 against anthrax has already been proven in previous studies (Korean Patent Registration No. 10-1765393).

Specifically, a suspension solution was prepared in which Bacillus Sirius NRS 731, an anthrax-like strain, was suspended in physiological saline so that the OD ₆₀₀ value was about 0.7. 0.1 ml of antibacterial protein B4 solution (test group) or physiological saline solution (control group) was added to the tube containing 0.9 ml of the prepared suspension. Thereafter, the OD ₆₀₀ value was measured for 20 minutes to examine the TOD ₅₀ value (the time taken to decrease to half of the initial bacterial absorbance).

At this time, the antibacterial protein B4 solution used was prepared and used as follows. A solution containing 1 mg/ml of antibacterial protein B4 was primarily prepared by adding 1 ml of water for injection to the lyophilized pharmaceutical composition prepared in Example 2, and water for injection was additionally added thereto to prevent antibacterial It was prepared by diluting so that the final concentration of protein B4 was 10 μg/ml.

Considering the structural characteristics, biological characteristics, and physical characteristics of the antimicrobial protein B4 as described above, several candidate formulations were devised, and an experiment to investigate the suitability thereof was repeatedly performed to obtain stable antimicrobial protein B4 in the same manner as in Example 2. A lyophilized pharmaceutical composition could be obtained. Specifically, it was prepared in the composition and process as shown in Table 1 below.

Item Contents Furtherance B4, 10 mM histidine, 0.1% (w/v) polysorbate 80, 5% (w/v) trehalose, pH 6.0 Freeze drying process Step 1) Freeze the solution to be lyophilized by storing it at -80℃ for one day
Step 2)
After placing the frozen sample in a freeze dryer, dry it at -20℃ for 16 hours.
Step 3)
After completion of “Step 2”, drying is continued for 4 hours while increasing by 10℃ every 1 hour (Temperature at final termination: 20℃)

Example 4. Confirmation of the excellence of the manufacturing method of the present invention

The excellence in the manufacturing process of the lyophilized composition having the final selected composition was confirmed as follows.

After applying the composition of Table 1, the purified fraction was lyophilized by the method of the present invention as it is, and a pharmaceutical composition prepared by the preparation method of the present invention by securing a lyophilized commercially available protein drug by the method of the present invention as a control. The excellence of the process was confirmed by comparing it with (hereinafter, the composition of the present invention).

The secured control group was a total of 3 types, and each control group 1 to 3 were as follows.

Control 1 is the purified fraction as it is lyophilized according to the lyophilization method of the present invention, Control 2 is a formulation applied to Nplate, a therapeutic agent for thrombocytopenia, among commercially available protein drugs (1.2 mg/ml histidine, 15 mg/ml sucrose, A composition to which 30 mg/ml mannitol, 0.03 mg/ml polysorbate 20, pH 6.0) was applied was lyophilized according to the lyophilization method of the present invention, and Control 3 was a formulation applied to Herceptin, an antibody drug among commercially available protein drugs ( It means that the composition to which 3.36 mg/ml histidine HCl, 2.16 mg/ml histidine, 136.2 mg/ml trehalose, 0.6 mg/ml polysorbate 20, pH 6.0) was applied was subjected to lyophilization according to the lyophilization method of the present invention. The effects of the present invention were confirmed by examining the stability of the control groups 1 to 3 and the compositions of the present invention secured as described above, respectively, at 37° C. for 12 weeks. At this time, the concentration of antibacterial protein B4 in the solution before lyophilization was 1 mg/ml.

The stability investigation of the control groups 1 to 3 and the composition of the present invention was performed through HPLC analysis, and the results were shown in FIG. 2. At this time, analysis was also conducted at week 0 in order to determine the initial value.

Figure 2 is a result of verifying the storage stability of the control group 1 to 3 and the composition of the present invention, showing a high-performance liquid chromatography analysis result, Figure 2 (a) is the analysis result of week 0 of the control group 1, (b) Is the analysis result of week 12 of control 1, (c) is the analysis result of week 0 of control 2, (d) is the analysis of week 12 of control 2, and (e) is analysis result of week 0 of control 3 , (f) shows the results of the 12 week analysis of the control group 3. Figure 2 (g) compared with these are the results of week 0 analysis of the composition of the present invention, and (h) is the results of week 12 analysis of the composition of the present invention.

The graph analysis results of FIG. 2 are summarized in Table 2 below.

Area value (relative area% compared to initial) Control 1 Control 2 Control 3 The present invention At week 0 12 weeks At week 0 12 weeks At week 0 12 weeks At week 0 12 weeks 2529988
(100%) 868599
(33.7%) 2575505
(100%) 1655154
(65.4%) 2598216
(100%) 1793141
(69.0%) 2440798
(100%) 2236196
(91.6%)

In addition, as a result of performing the investigation of the biological activity, an analysis result as shown in FIG. 3 was shown. Similar to the experiment of FIG. 2, analysis was also performed at the time of week 0 to determine the initial value.

Figure 3 shows the results of analyzing the biological activity of the composition of the present invention compared to the control group 1 to 3, Figure 3 (a) is the analysis result of week 0 of the control 1, (b) is the analysis of week 12 of the control 1 Results are shown, (c) is the analysis result of week 0 of control 2, (d) is the analysis result of week 12 of control 2, (e) is the analysis result of week 0 of control 3, (f) is control 3 It shows the results of the 12th week analysis. Figure 3 (g) compared with these are the results of week 0 analysis of the composition of the present invention, and (h) is the results of week 12 analysis of the composition of the present invention. In the graph of FIG. 3, (+) indicates the case of adding the antibacterial protein B4 solution, and (-) indicates the case of adding physiological saline instead of the antibacterial protein B4 solution as a negative control.

The results of analyzing the graph of FIG. 3 are summarized in Table 3 below. Looking at Table 3, it can be seen that the TOD ₅₀ value was not at all derived at the time point of 12 weeks for the control groups 1 to 3, whereas the composition of the present invention maintained almost the same value as the initial value at the time point of 12 weeks. Through these results, in the case of the composition prepared by the production method of the present invention, it could be proved that the biological activity is not reduced for 12 weeks and can be stably stored.

TOD ₅₀ (minutes) Control 1 Control 2 Control 3 The present invention At week 0 12 weeks At week 0 12 weeks At week 0 12 weeks At week 0 12 weeks 2.92 Not derived 2.53 Not derived 2.66 Not derived 2.50 2.70

As can be seen from the above results, the pharmaceutical composition having an anthrax-specific antimicrobial activity prepared using the preparation method of the present invention or the composition of the present invention is very stable in storage at 37°C for 12 weeks and has antibacterial activity. It was confirmed that it was well maintained even after storage at 37°C for 12 weeks.

That is, it was verified that the pharmaceutical composition of the freeze-dried formulation containing the anthrax-specific antibacterial protein B4 of the present invention is suitable for realizing the above-described effects due to high storage stability as well as possible storage at room temperature.

As described above, although the embodiments have been described by the limited drawings, a person of ordinary skill in the art can apply various technical modifications and variations based on the above. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a form different from the described method, or are replaced or substituted by other components or equivalents. Appropriate results can be achieved.

Therefore, other implementations, other embodiments and claims and equivalents fall within the scope of the following claims.

Korea Research Institute of Bioscience and Biotechnology KCTC12709BP 20141112

<110> Agency for Defense Development <120> Pharmaceutical composition having anthrax-specific antimicrobial activity and method for producing the same <130> APC-2018-0519 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 262 <212> PRT <213> Artificial Sequence <220> <223> Antibacterial protein having lytic activity specific to Bacillus anthracis from deposited Escherichia coli <400> 1 Met Ala Met Ala Leu Gln Thr Leu Ile Asp Lys Ala Asn Arg Lys Leu 1 5 10 15 Asn Val Ser Gly Met Arg Lys Asp Val Ala Asp Arg Thr Arg Ala Val 20 25 30 Ile Thr Gln Met His Ala Gln Gly Ile Tyr Ile Cys Val Ala Gln Gly 35 40 45 Phe Arg Ser Phe Ala Glu Gln Asn Ala Leu Tyr Ala Gln Gly Arg Thr 50 55 60 Lys Pro Gly Ser Ile Val Thr Asn Ala Arg Gly Gly Gln Ser Asn His 65 70 75 80 Asn Tyr Gly Val Ala Val Asp Leu Cys Leu Tyr Thr Gln Asp Gly Ser 85 90 95 Asp Val Ile Trp Thr Val Glu Gly Asn Phe Arg Lys Val Ile Ala Ala 100 105 110 Met Lys Ala Gln Gly Phe Lys Trp Gly Gly Asp Trp Val Ser Phe Lys 115 120 125 Asp Tyr Pro His Phe Glu Leu Tyr Asp Val Val Gly Gly Gln Lys Pro 130 135 140 Pro Ala Asp Asn Gly Gly Ala Val Asp Asn Gly Gly Gly Ser Gly Ser 145 150 155 160 Thr Gly Gly Ser Gly Gly Gly Ser Thr Gly Gly Gly Ser Thr Gly Gly 165 170 175 Gly Tyr Asp Ser Ser Trp Phe Thr Lys Glu Thr Gly Thr Phe Val Thr 180 185 190 Asn Thr Ser Ile Lys Leu Arg Thr Ala Pro Phe Thr Ser Ala Asp Val 195 200 205 Ile Ala Thr Leu Pro Ala Gly Ser Pro Val Asn Tyr Asn Gly Phe Gly 210 215 220 Ile Glu Tyr Asp Gly Tyr Val Trp Ile Arg Gln Pro Arg Ser Asn Gly 225 230 235 240 Tyr Gly Tyr Leu Ala Thr Gly Glu Ser Lys Gly Gly Lys Arg Gln Asn 245 250 255 Tyr Trp Gly Thr Phe Lys 260

Claims (10)

As a method for preparing a pharmaceutical composition having specific antibacterial activity for anthrax,
Mixing anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1 and a stabilizing solution;
Freezing the mixed mixture;
First drying the frozen mixture; And
Drying the first dried mixture by repeating at least two times;
Including,
The step of drying by repeating the above two or more times is a step of repeating drying while increasing the drying temperature,
The pharmaceutical composition having specific antibacterial activity for anthrax bacteria,
Anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1; And
Stabilizing solution; Including,
The stabilizing solution,
Histidine (L-Histidine) having a concentration of 1 mM to 50 mM;
Polysorbate 80 with a concentration of 0.05% (w/v) to 10% (w/v) and
A method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity, comprising trehalose having a concentration of 1% (w/v) to 10% (w/v). delete The method of claim 1,
The stabilizing solution is a solution of pH 5.0 to pH 9.0, a method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity. The method of claim 1,
The freezing is carried out in a temperature range of -90°C to -70°C, a method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity. The method of claim 1,
The first drying is carried out in a temperature range of -30°C to -10°C, a method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity. The method of claim 1,
Obtaining an anthrax antibacterial protein B4 from Escherichia coli BL21-pBAD-B4 strain [accession number: KCTC 12709BP]; A method for preparing a pharmaceutical composition having an anthrax-specific antibacterial activity further comprising a. Anthrax-specific antibacterial protein B4 represented by SEQ ID NO: 1; And
Stabilizing solution; Including,
The stabilizing solution,
Histidine (L-Histidine) having a concentration of 1 mM to 50 mM;
Polysorbate 80 with a concentration of 0.05% (w/v) to 10% (w/v) and
A pharmaceutical composition having an anthrax-specific antibacterial activity, comprising trehalose having a concentration of 1% (w/v) to 10% (w/v). The method of claim 7,
The stabilizing solution is a pharmaceutical composition having a specific antibacterial activity of pH 5.0 to pH 9.0. The method of claim 7,
The anthrax-specific antibacterial protein B4 is obtained from the Escherichia coli BL21-pBAD-B4 strain [accession number: KCTC 12709BP], a pharmaceutical composition having an anthrax-specific antibacterial activity. A drug having an anthrax-specific antibacterial activity comprising the composition of any one of claims 7 to 9.

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