AU2018310071B2 - Method for obtaining Bordetella pertussis-derived protein, comprising freezing and thawing process - Google Patents

Abstract

The present invention relates to a method for obtaining a

Description

Description

Title of Invention METHOD FOR OBTAINING BORDETELLA PERTUSSIS-DERIVED PROTEIN, COMPRISING FREEZING AND THAWING PROCESS

Technical Field

The present invention relates to a method for purifying Bordetella pertussis derived PRN protein, comprising a freezing and thawing process.

Background Art

Pertussis is an acute respiratory disease that occurs mainly in infants and is characterized by coughing of 2 weeks or longer. Bordetella pertussis, which is a Gram-negative, aerobic, short coccobacillus, has been reported to cause pertussis. Bordetella pertussis uses humans as its only host and is infected mainly through the respiratory tract. In addition, Bordetella pertussis lives on the respiratory tract mucosa and causes a disease in the human body. In the 1930s, a cellular pertussis vaccine was developed, and this vaccine proved to have a prophylactic effect on pertussis. In addition, pertussis vaccines were used in combination with tetanus and diphtheria inactivated killed vaccines in the 1940s; however, adverse effects (seizure, edema, fever, and the like) of whole-cell pertussis vaccines were reported. Thus, there was a need to develop a pertussis vaccine with safety.

In the 1950s, studies on pathogenesis of Bordetella pertussis were carried out, and components such as pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM) were reported as antigens. Then, development of acellular pertussis vaccines, which involves isolation and purification of these proteins, was underway. After the 1980s, a purified pertussis vaccine was developed and vaccinated for the first time in Japan.

Proteins such as PT, FHA, and PRN have to be purified to prepare acellular pertussis vaccines. Traditionally, the antigens were purified simultaneously by repeating ammonium sulfate precipitation and density gradient centrifugation. However, such a method has disadvantages that a lot of impurities are generated and it is difficult to control the purification process. Another method is to purify each antigen individually using a combination of physical and chemical methods. Korean Laid-Open Patent Publication No. 2015-0124973 discloses an acellular pertussis vaccine composition containing PT, FHA, and FIM types 2 and 3.

Meanwhile, in a pertussis vaccine production method which comprises mixing three or four components, there is a problem that decreased productivity of even one component may increase the overall production period.

Therefore, a method for efficiently producing PRN protein in large quantities is being sought to efficiently prepare a pertussis vaccine.

Any discussion of documents, acts, materials, devices, articles of the likes which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

Disclosure of Invention

Regarding this, while making efforts to obtain conditions that can result in an increased extraction amount of PRN protein of Bordetella pertussis, the present inventors have identified that in a case where a sample containing Bordetella pertussis undergoes freezing (cold-shock) and thawing steps under specific conditions, an extraction amount of PRN protein of Bordetella pertussis is increased, thereby completing the present invention.

Accordingly, in one broad form, the present disclosure provides a method for obtaining PRN protein which can result in an increased extraction amount of PRN protein of Bordetella pertussis.

In a particular form, the present disclosure provides a method for obtaining Bordetella pertussis-derived PRN protein, comprising the steps of: 1) freezing a sample containing Bordetella pertussis; 2) thawing the frozen sample; 3) disrupting the thawed sample; and 4) purifying the disrupted sample.

In one particular aspect, the present disclosure provides a method for obtaining Bordetella pertussis-derivedPRN protein, comprising the steps of:

1) freezing a sample containing Bordetella pertussis;

2) thawing the frozen sample;

3) disrupting the thawed sample; and

4) purifying the disrupted sample,

wherein in step 2), the thawing is carried out in refrigeration at a temperature is 1°C to 0C for 12 hours to 60 hours.

The method for obtaining PRN protein of Bordetellapertussis according to the present invention suitably comprises freezing a sample containing Bordetella pertussis and thawing the sample at a refrigeration temperature, so that an extraction amount of the PRN protein is maximized. A remarkably increased extraction amount of PRN protein was obtained in a case where the PRN protein is obtained by purifying a sample that has been subjected to pretreatment under the above condition, as compared with an extraction method that does not involve such pretreatment. In addition, the method for obtaining PRN protein can also be effectively used for mass production of PRN protein for the purpose of producing pertussis vaccines.

Brief Description of Drawings

FIG. 1 illustrates a flowchart of PRN purification process.

FIG. 2 illustrates a detailed flowchart of process for pellet preparation and thawing and process for treatment with urea.

FIG. 3 illustrates results obtained by subjecting each of pellets, which have been obtained under varying preparation and storage conditions, to treatment with urea.

FIG. 4A illustrates, using SDS-PAGE technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 1to 5 and Examples 1 to 3.

FIG. 4B illustrates, using SDS-PAGE technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 6 to 10, and Examples 4 and 5.

FIG. 4C illustrates, using SDS-PAGE technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 11 to 16 and Example 6.

FIG. 5A illustrates, using Western blotting technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 1 to 5 and Examples 1 to 3.

FIG. 5B illustrates, using Western blotting technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 6 to 10 and Examples 4 and 5.

FIG. 5C illustrates, using Western blotting technique, difference in extraction amount of PNR protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 11 to 16 and Example 6.

FIG. 6 illustrates an extraction amount of PRN protein in a case where the PRN protein is obtained by each of the methods of Comparative Examples 1 to 7 and Examples 1 to 3 with varying slurry and pellet storage conditions.

FIG. 7 illustrates an extraction amount of PRN protein in a case where the

PRN protein is obtained by each of the methods of Comparative Examples 15 to 17 and Example 7 with varying pellet storage conditions.

FIG. 8 illustrates difference in extraction amount of PNR protein in a case where varying pellet thawing conditions are used.

Detailed description

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

In an aspect of the present invention, there is provided a method for obtaining Bordetella pertussis-derived PRN protein, comprising the steps of: 1) freezing a sample containing Bordetella pertussis; 2) thawing the frozen sample; 3) disrupting the thawed sample; and 4) purifying the disrupted sample.

First, a step of freezing a sample containing Bordetellapertussis is carried out.

In the present invention, "Bordetella pertussis" is also called Bordetella parapertussis, and is a Gram-negative coccobacillus as small as approximately 0.3 to 1 m, without flagella and spores. PRN, one of the main proteins of Bordetella pertussis, is an abbreviation of pertactin and a virulence factor of Bordetellapertussis that causes pertussis. In addition, PRN, an outer membrane protein that adheres to tracheal epithelial cells, is obtained from Bordetella pertussis and is one of the important components of pertussis vaccines.

In the present specification, the "sample containing Bordetella pertussis" may be a culture obtained by culturing Bordetella pertussis, slurry separated from the culture through serial centrifugation, or a pellet obtained from the slurry through high speed centrifugation, with the pellet being preferred. Here, the obtained slurry may be immediately centrifuged to obtain a pellet. However, the obtained slurry may be centrifuged after 1 to 5 days, to obtain a pellet. Here, the slurry may be stored at 0°C to 25°C. However, the present invention is not limited thereto.

In an embodiment of the present invention, a pellet was prepared by culturing a Bordetella pertussis strain in modified stainer scholte (MSS) medium at a temperature of 35°C, subjecting the cell culture to serial centrifugation for 2 hours at room temperature, and subjecting the slurry obtained therefrom to high-speed centrifugation at a speed of 7,000 rpm for 50 minutes at 2°C to 8°C.

Secondly, a step of thawing the frozen sample is carried out.

In the step of freezing the sample containing the Bordetella pertussis strain, the freezing may be carried out at a temperature of -1°C,-2°C, -5°C, -10C, -200 C, 0C, -40C, -50C, -60C, -70°C, -80°C, or -90°C, or at a temperature of -1C to °C, -5°C to -85°C, -15°C to -75°C, or -30°C to -60°C. The freezing may be carried out for 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 20 hours, 30 hours, or 40 hours, or for 0.5 hours to 40 hours, 1 hour to 30 hours, 2 hours to 20 hours, or 5 hours to 10 hours.

In addition, the frozen sample is slowly thawed by being stored refrigerated. Here, the thawing may be carried out by being refrigerated at a temperature of1°C, 2 0C, 3 0C, 4 0C, 5 0C, 7 0C, 10C, 15C, 20C, or 25C, or at a temperature of 1°C to 25C, 2 0C to 180 C, 50 C to 15 0 C, or 70 C to10°C. The thawing may be carried out by being refrigerated for 0.1 hours, 1 hour, 5 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, or 60 hours, or for 0.1 hour to 60 hours, 1 hour to 55 hours, 5 hours to 50 hours, or 12 to 48 hours.

In an embodiment of the present invention, the pellet was stored frozen at -300 C or lower for 2 hours, and the frozen pellet was thawed at room temperature or a refrigeration temperature of 1°C to 7C.

Here, the freezing step and the thawing step may be repeatedly performed one or more times.

Thirdly, a step of disrupting the thawed sample is carried out.

The thawed sample is subjected to the disrupting step. Here, the disrupting may be carried out through, for example, treatment with urea, thermal treatment, treatment with guanidine, and treatment with iodine. However, the disrupting method is not limited thereto and various disrupting methods known in the art may be applied.

In a case where the sample is disrupted by treatment with urea, the treatment may be performed at a urea concentration of 0.1 M, 0.5 M, 1M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, or 10 M, or at a urea concentration of 0.1 M to 10 M, 1 M to 8 M, 3 M to 7 M, or 4 M to 5 M. The treatment with urea may be performed for 5 minutes, 10 minutes, 20 minutes, 50 minutes, 80 minutes, 120 minutes, 180 minutes, 240 minutes, or 360 minutes, or for 5 minutes to 360 minutes, 20 minutes to 240 minutes, or 50 minutes to 180 minutes.

In an embodiment of the present invention, the outer membrane of Bordetella pertussis in a pellet was disrupted by subjecting the thawed pellet to 5.0 M urea buffer in an amount 4 times the pellet weight, and performing stirring for 3 hours.

Fourthly, a step of purifying the disrupted sample is carried out.

After centrifuging a cell lysate or cell culture, unnecessary cell debris may be removed using various column chromatography methods and the like. The column chromatography method may include ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase-HPLC, affinity chromatography, and the like.

In an embodiment of the present invention, in the step of purifying the disrupted pellet, PRN protein was obtained by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration chromatography (GFC).

As used herein, the "ion exchange chromatography" may use an ion exchange resin in which anions or cations are covalently bonded to the stationary phase which is an exchange resin. Oppositely charged solute ions in the mobile phase are attracted to the stationary phase by electrostatic attraction. Ion exchange chromatography is based on equilibrium achieved by adsorption of ions or electrically charged compounds onto an ion exchanger due to electrostatic force.

In an embodiment of the present invention, PRN protein was eluted by using an anion exchange resin in an ion exchange chromatography column, and using Tris HCl solution as equilibration buffer, Tris-HCl with NaCl solution as washing buffer, and Tris-HCl with NaCl solution as elution buffer.

As used herein, the term "hydrophobic interaction chromatography" refers to a separation method using hydrophobic interaction between a matrix having a hydrophobic functional group and a molecule. The matrix is allowed to have hydrophobic functionality by modification of agarose that is hydrophilic and inactive. Modified agarose obtained by reacting alkylamine with BrCN-activated agarose is widely used. Hydrophobic interaction chromatography is widely used for protein pure separation. In addition, in hydrophobic interaction chromatography, for protein elution, ionic strength may be decreased or pH may be increased, and aliphatic amines that decrease polarity, alcohols, or nonionic detergents (for example, Tween-20 and Triton X-100) may be used.

In an embodiment of the present invention, PRN protein was eluted by using Tris-HCl solution with NaCl solution as equilibration buffer, Tris-HCl solution with NaCl solution as washing buffer, and Tris-HCl solution with NaCl solution as elution buffer.

As used herein, the "gel filtration chromatography" uses molecular sieves in the stationary phase, the molecular sieves being Sephadex, polyacrylamide, or agarose gels which are hydrophilic and thus are capable of absorbing water so as to be swelled. In a case where a sample molecule has a size larger than the maximum pore of the swollen gel, the molecule does not pass through the gel particles and thus exits a column through space in stationary phase particles. Smaller molecules enter open pores of the gel particles, and pass therethrough at varying rates depending on their size and shape. Thus, the molecules are eluted in order of their decreasing size. In gel chromatography, sample size, viscosity, ionic strength, flow rate, and the like are considered.

In an embodiment of the present invention, PRN protein was eluted using sodium phosphate with NaCl solution as equilibration buffer.

Mode for the Invention

Hereinafter, the present invention will be described in more detail by way of the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited only thereto.

Example 1. Process of obtaining PRN

The process of obtaining PRN was carried out in the following process steps: pellet preparation and thawing, treatment with urea, column chromatography (IEX, HIC, and SEC), concentration and buffer exchange (UF/DF), and purification of impurity-free PRN protein. A flowchart of the process of obtaining PRN is shown in FIG. 1.

Example 1.1. Culture of Bordetella pertussis

Bordetella pertussis (Korea National Institute of Health) was cultured in MSS medium at a temperature of 35°C for 4 days. For the culture period, specifically, it took 1 day for seed culture, 1 day for primary enrichment culture, 1 day for secondary enrichment culture, and 1 day for main culture.

Example 1.2. Pellet preparation

The cell culture was separated into culture supernatant and slurry by serial centrifugation under a condition of a flow rate of 100 L/h and 9,500 rpm for 2 hours at room temperature. The separated slurry was subjected to high-speed centrifugation at 7,000 rpm for 50 minutes at 5°C ±3C, to prepare a pellet. In the following examples, in a case where the slurry is stored for 3 days, the slurry was stored for 3 days at a refrigeration temperature of 5°C, and then subjected to high-speed centrifugation, to prepare a pellet.

Example 1.3. Freezing and thawing

The prepared pellet was stored at -30°C or lower for 2 hours, and then thawed by being stored at room temperature (RT) for 1 day. Subsequently, PRN protein was obtained by performing procedures of following Examples 1.4 to 1.7. In addition, experiments were conducted with varying storage, and freezing and thawing conditions of the slurry as in Examples 1 to 6 and Comparative Examples 1 to 16 shown in Table 1 below, and then PRN protein was obtained by performing procedures of following Examples 1.4 to 1.7.

[Table 1] ExperimentNo. Comp Exam Compara Exam Exam Compara Compara Compara Compara Compara Compar arative ple1 five ple2 ple3 five tive five tive tive active Examp Example Example Example Example Example Example Example le1 2 3 4 5 6 7 8

Slurr Pellet Cold- X 0 X 0 0 X X X X X X y, storage shock stor conditi ed on Storage X Room temperature Refrigeration (Cold room) Frozen 1 Frozen 2 for condition (RT) 0 (F1, -30°C) (F2,-70°C)

Storage X 1 1 1 3 1 3 1 3 1 3 days ExperimentNo. Comp Exam Compara Exam Exam Compara Compara Compara Compara Compara Compar arative ple4 five ple5 ple6 five tive five tive tive active Examp Example Example Example Example Example Example Example le9 10 11 12 13 14 15 16

Slurr Pellet Cold- X 0 X 0 0 X X X X X X y, storage shock stor conditi ed on Storage X Room temperature Refrigeration (Cold room) Frozen 1 Frozen 2 for condition (RT) 3 (F1, -30°C) (F2,-70°C) day orange X 1 1 1 3 1 3 1 3 1 3 Days Example 1.4. Treatment with urea

To the thawed pellet was added 5.0 M urea buffer in an amount 4 times the pellet weight; and stirring was performed for 3 hours at a speed of 280 rpm using a magnetic bar. The urea-treated solution, which had been stirred for 3 hours, was subjected to high-speed centrifugation under a condition of 5°C 3°C for 1.5 hours at a speed of 7,000 rpm (12230 RCF). After the centrifugation, cell debris was removed and the supernatant was collected. The collected supernatant was filtered through a 0.22 pm filter. A detailed process flowchart of the pellet preparation and the thawing of Examples 1.2 and 1.3, and the treatment with urea is illustrated in FIG. 2.

Example 1.5. Purification on IEX column

Distilled water (DW) was flowed, in 3 CVs at 100 cm/hr, through a column filled with IEX resin, to remove the storage solution. Then, a IN NaOH solution was flowed therethrough at 40 cm/hr for 1 hour, to perform washing. Tris-HCl solution, which is equilibration buffer, was flowed therethrough in 3 CVs, to achieve equilibrium. After completion of the equilibrium, a process solution for concentration and buffer exchange 1 was flowed through the IEX column for adsorption. The equilibration buffer was flowed therethrough in 3 CVs, to remove the solution remaining in the column. Subsequently, Tris-HCl solution with NaCl solution, which is washing buffer, was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, Tris-HCl solution with NaCl solution, which is elution buffer, was flowed therethrough in 5 CVs, to elute PRN protein.

Example 1.6. Purification on HIC column

Distilled water was flowed, in 3 CVs at 100 cm/hr, through a column filled with HIC resin, to remove the storage solution. Then, a IN NaOH solution was flowed therethrough at 40 cm/hr for 1 hour, to perform washing. Tris-HCl solution with NaCl solution, which is equilibration buffer, was flowed therethrough in 3 CVs, to achieve equilibrium. After completion of the equilibration, a solution, which had been obtained by mixing the eluate obtained through the purification process on the IEX column with 3.6 M NaCl in a 1:1 ratio, was flowed through the HIC column for adsorption. The equilibration buffer was flowed therethrough in 3 CVs to remove the solution remaining in the column. Subsequently, Tris-HCl solution with NaCl solution, which is washing buffer, was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, Tris-HCl solution with NaCl solution, which is elution buffer, was flowed therethrough in 5 CVs, to elute PRN protein.

Example 1.7. Purification on GFC column

Distilled water was flowed, in 3 CVs at 15 cm/hr, through a column filled with GFC resin, to remove the storage solution. Then, a IN NaOH solution was flowed therethrough at 15 cm/hr for 1 hour, to perform washing. Sodium phosphate with NaCl solution, which is equilibration buffer, was flowed therethrough in 3 CVs, to achieve equilibrium. After completion of the equilibrium, a process solution for concentration and buffer exchange 2 was flowed through the GFC column, to elute PRN protein.

Experimental Example 1. Identification of difference in PRN extraction amount depending on pellet preparation and storage conditions

In order to identify conditions under which an extraction amount of PRN protein is effectively increased, experiments were conducted with varying pellet preparation and storage conditions in the process of obtaining PRN of Example 1. The pellet preparation and storage conditions are as shown in Table 1 of Example 1.3, and experimental procedures and results depending on the respective conditions are shown in following Experimental Examples 1.1 to 1.5.

Experimental Example 1.1. Changes in supernatant color due to treatment with urea depending on slurry and pellet storage conditions

Each of pellets, obtained by using varying preparation and storage conditions as shown in Table 1 of Example 1.3, was subjected to treatment with urea buffer, and stirring was performed. Then, the resultant was centrifuged. The supernatant obtained through the centrifugation was filtered with a filter, and color difference of the supernatant depending on each storage condition was identified. The above process is described in detail in Example 1.4 and the experimental results are illustrated in FIG. 3.

As illustrated in FIG. 3, the supernatant showed dark yellow color at the conditions (Examples 2 and 3, and Examples 5 and 6) under which the pellet was stored frozen and then stored refrigerated. This means that many pellets are broken in a case of being subjected to treatment with urea after being stored frozen and thawed in refrigeration.

Experimental Example 1.2. Identification of difference in protein extraction amount depending on pellet storage conditions

SDS-PAGE and Western blotting techniques were used to identify difference in extraction amount of PRN protein depending on pellet storage conditions. Here, in the Western blotting technique, Guinea Pig anti-PRN (Young In Frontier) and Biotin-labeled Guinea Pig anti-PRN (Young In Frontier) were used as antibodies. These results are illustrated in FIGS. 4 and 5.

As illustrated in FIGS. 4 and 5, a large amount of PRN protein was obtained at the conditions (Examples 1 to 6) under which the pellet was stored frozen and then stored refrigerated.

Experimental Example 1.3. Identification of extraction amount of PRN protein depending on slurry and pellet storage conditions

Enzyme-linked immunosorbent assay (ELISA) technique was used to identify difference in extraction amount of PRN protein depending on slurry and pellet storage conditions. These results are illustrated in FIG. 6. Here, the respective pellet storage conditions for item Nos. 1to 11 at the bottom of the graph in FIG. 6 are as follows, and the more detailed conditions are shown in Table 2 below:

1: Treated with urea immediately after pellet preparation;

2: stored frozen and thawed at room temperature for 1 day;

3: thawed at room temperature for 1 day without being stored frozen;

4: stored frozen and thawed in refrigeration for 1 day;

5: stored frozen and thawed in refrigeration for 3 days;

6: thawed in refrigeration for 1 day without being stored frozen;

7: thawed in refrigeration for 3 days without being stored frozen;

8: stored frozen (-30°C) for 1 day;

9: stored frozen (-30°C) for 3 days;

10: stored frozen (-70°C) for 1 day; and

11: stored frozen (-70°C) for 3 days.

[Table 2] Slurry, stored for 0 day Slurry, stored for 3 days

1. Treated immediately after pellet Comparative Example 1 Comparative Example 9 preparation

2. Stored frozen (C.S) and then stored at Example 1 Example 4 RT for 1 day

3. Stored at RT for 1 day Comparative Example 2 Comparative Example 10

4. Stored frozen (C.S) and then stored Example 2 Example 5 refrigerated for 1 day

5. Stored frozen (C.S) and then stored Example 3 Example 6 refrigerated for 3 days

6. Stored refrigerated for 1 day Comparative Example 3 Comparative Example 11

7. Stored refrigerated for 3 days Comparative Example 4 Comparative Example 12

8. Stored frozen 1 (-30°C) for 1 day Comparative Example 5 Comparative Example 13

9. Stored frozen 1 (-30°C) for 3 days Comparative Example 6 Comparative Example 14

10. Stored frozen 2 (-70°C) for 1 day Comparative Example 7 Comparative Example 15

11. Stored frozen 2 (-70°C) for 3 days Comparative Example 8 Comparative Example 16

As illustrated in FIG. 6, the pellet, which had been prepared after being stored for 0 day in a slurry state, exhibited an increase in extraction amount of PRN protein over time during which the pellet is stored frozen (by comparison between Examples 2 and 3); and the pellet, which had been prepared after being stored for 3 days in a slurry state, exhibited a decrease in extraction amount of PRN protein over time during which the pellet is stored frozen (by comparison between Examples 5 and 6). In addition, the pellet having undergone frozen storage exhibited an increase in extraction amount of PRN protein as compared with the pellet having not undergone frozen storage (by comparison between Example 1 and Comparative Example 2, comparison between Example 2 and Comparative Example 3, and comparison between Example 3 and Comparative Example 4). This means that in order to increase an extraction amount of PRN protein, it is more effective to immediately pelletize slurry and store the pellet than to store the slurry as it is, and that the freezing step causes a large increase in extraction amount of PRN protein.

Experimental Example 1.4. Identification of difference in extraction amount of PRN protein depending on pellet thawing

In FIG. 6, among experimental groups (Examples 1 and 4, Examples 2 and 5, Examples 3 and 6, Comparative Examples 5 and 13, Comparative Examples 6 and 14, Comparative Examples 7 and 15, and Comparative Examples 8 and 16) which had been stored frozen, the experimental groups (Examples 1 and 4, Examples 2 and 5, and Examples 3 and 6), which had been stored frozen and then undergone thawing, exhibited a remarkable increase in extraction amount of PRN protein. Therefore, further experiments were conducted to identify whether an extraction amount of PRN protein varies depending on thawing. Here, pellet thawing was performed for 48 hours at a refrigeration temperature of 5°C. The experimental results are shown in Table 3 and FIG. 7.

[Table 3] Condition PRN Conc. Detailed storage condition Pellet state before No. treatment with urea (pg/mL)

Comparative 39.95 Slurry, stored for 3 days; pellet, stored for 1 day at -70°C Frozen Example 15

Comparative 34.68 Slurry, stored for 3 days; pellet, stored for 3 days at -70°C Frozen Example 16

Comparative 32.31 Slurry, stored for 3 days; pellet, stored for 10 days at -70°C Frozen Example 17

Example 7 99.94 Slurry, stored for 3 days; pellet, stored for 10 days at -70°C Thawed (5°C, 48 hours)

As shown in Table 3 and FIG. 7, in a case where the pellet is thawed before treatment with urea, an approximately 3-fold increase in extraction amount of PRN protein was observed as compared with a case where the pellet is frozen. In all three experimental groups in which the pellet frozen is subjected to treatment with urea, the PRN protein was extracted at a low level. This means that pellet thawing before treatment with urea has a great effect on PRN protein extraction. From these results, it was found that the pellet has to be thawed before treatment with urea in order to effectively increase an extraction amount of PRN protein.

Experimental Example 1.5. Identification of difference in extraction amount of PRN protein depending on pellet thawing conditions

In order to identify difference in extraction amount of PRN protein depending on pellet thawing methods, an extraction amount of PRN protein was checked in a case where the pellet is stored frozen and then stored refrigerated (for 2 days), at room temperature (for 1 hour), and at 37C (for 15 minutes), respectively. These results are illustrated in FIG. 8.

As illustrated in FIG. 8, when thawing is performed, an increased extraction amount of PRN protein was observed in a case of being slowly thawed in refrigeration as compared with a case of being rapidly thawed at a high temperature. In addition, an approximately 2-fold increase in extraction amount of PRN protein was observed in a case of being thawed for 2 days in refrigeration as compared with a case of being thawed for 15 minutes at 37°C. From these results, it was found that the pellet has to be slowly thawed in refrigeration after being frozen in order to effectively increase an extraction amount of PRN protein.

Experimental Example 2. Identification of scale-up possibility

In order to identify whether the conditions identified in Experimental Examples 1.1 to 1.5, under which an extraction amount of PRN protein is effectively increased, can be applied even at a 50-L scale, the slurry was immediately pelletized without being stored, and the resulting pellet was stored frozen (-30°C) for 1 day and then thawed in refrigeration (5°C) for 2 days. Subsequently, 533 g of pellet was collected, subjected to treatment with 1,800 mL of 5.0 M urea buffer, and stirred for 3 hours using a magnetic bar. The stirred urea-treated solution was centrifuged for 1.5 hours at a speed of 6,300 rpm. After the centrifugation, cell debris was removed; and 1,650 mL of the supernatant was collected and filtered through a filter. Subsequently, the procedures in Examples 1.5 to 1.7 were performed. The results are shown in Table 4.

[Table 4] Total volume (mL) PRN Conc. (pg/mL) Total PRN amount (mg)

PRN 176.3 701.98 123.76

As shown in Table 4, it was found that the conditions identified in Experimental Examples 1.1 to 1.5, under which an extraction amount of PRN protein is effectively increased, can be applied even at a 50-L scale, and that about 300 mg to 400 mg of PRN protein can be obtained at a 200-L scale.

Claims (11)

Claims [Claim 1] A method for obtaining Bordetella pertussis-derived PRN protein, comprising the steps of:

1) freezing a sample containing Bordetella pertussis;

2) thawing the frozen sample;

3) disrupting the thawed sample; and

4) purifying the disrupted sample,

wherein in step 2), the thawing is carried out in refrigeration at a temperature is 1°C to 0C for 12 hours to 60 hours.

[Claim 2]

The method of claim 1, wherein the sample containing Bordetellapertussis is a pellet obtained by centrifuging a Bordetella pertussis culture.

[Claim 3]

The method of claim 1, wherein in step 1), the freezing is carried out at a temperature of -50 C to -850 C.

[Claim 4]

The method of claim 1, wherein in step 1), the freezing is carried out for 20 to hours.

[Claim 5]

The method of claim 1, wherein in step 2), the thawing is carried out in refrigeration.

[Claim 6]

The method of claim 5, wherein the thawing is carried out by being refrigerated at a temperature of 1°C to 10°C.

[Claim 7]

The method of claim 5, wherein the thawing is carried out by being refrigerated for 36 to 48 hours.

[Claim 8]

The method of claim 1, wherein in step 3), the disrupting is carried out through any one selected from the group consisting of treatment with urea, thermal treatment, treatment with guanidine, and treatment with iodine.

[Claim 9]

The method of claim 8, wherein the treatment with urea is performed at a urea concentration of 3 M to 7 M.

[Claim 10]

The method of claim 8, wherein the treatment with urea is performed for 150 to 210 minutes.

[Claim 11]

The method of claim 1, wherein in step 4), the purifying is carried out by performing ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration chromatography (GFC).