AU2019242986A1 - Bordetella pertussis-derived protein-obtaining method including affinity chromatography process - Google Patents

Abstract

The present invention relates to a Bordetella pertussis-derived protein-obtaining method including an affinity chromatography process. The present invention increases the production yield of PT and FHA proteins by isolating PT and FHA proteins of Bordetella pertussis through a purification process using a blue affinity column. In addition, when used, the filtration process can remarkably increase the output of the target proteins, compared to an isolation method using a size exclusion chromatography (SEC) process. Furthermore, the method using the blue affinity column for isolation of PT and FHA proteins decreased resin expense, process time, buffer consumption, and production cost and significantly increased the productivity of PT and FHA proteins.

Description

Description

Title of Invention BORDETELLA PERTUSSIS-DERIVED PROTEIN-OBTAINING METHOD INCLUDING AFFINITY CHROMATOGRAPHY PROCESS

Technical Field The present invention relates to a method for obtaining a Bordetella Pertussis derived protein, including an affinity chromatography process.

Background Art Pertussis is an acute respiratory disease that occurs mainly in infants and is characterized by coughing for 2 weeks or longer. Bordetella pertussis, which is a Gram-negative, aerobic, short coccobacillus, has been reported to cause pertussis. Bordetellapertussis uses human as its only host and human 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 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. Since 1980s, a purified pertussis vaccine was developed and vaccinated for the first time in Japan. The purpose of purification in pertussis vaccines is to produce products that are rich in specific proteins such as PT, FHA, and PRN and are free of endotoxin.

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. The existing process for purifying PT and FHA, which are the main proteins of Bordetella Pertussis, is to isolate PT and FHA present in a culture supernatant, using size exclusion chromatography (SEC), a method used for common vaccine preparations. However, the method is difficult to scale up and has a low process yield. In addition, in a pertussis vaccine production method which comprises mixing three or four components, decreased productivity of even one component may result in a problem of increasing the overall production period as well as an economical problem caused by increased number of production batches.

Disclosure of Invention Technical Problem Regarding this, while studying to solve the problems with the SEC process and to find a method that enables efficient production of PT and FHA which are Bordetella Pertussis-derivedproteins, the present inventors have found that in a case where PT and FHA proteins are isolated through affinity chromatography using a resin to which a specific compound is bound, not only the time and cost required for the process are greatly decreased, but also the production of the target proteins is increased. Based on this finding, the present inventors have completed the present invention. Accordingly, an object of the present invention is to provide a method for isolating PT protein or FHA protein, which is a Bordetella Pertussis-derivedprotein, using an affinity column. Another object of the present invention is to provide a method for purifying PT protein or FHA protein which is a Bordetella Pertussis-derivedprotein.

Solution to Problem In order to achieve the above object, the present invention provides a method for isolating, through an affinity chromatography process, PT protein or FHA protein in a pre-treated Bordetella Pertussis culture. In order to achieve the above object, the present invention provides a method for purifying PT protein, comprising a step of removing, through a membrane chromatography (MC) process, endotoxin from the PT protein that has been isolated in the affinity chromatography process. In addition, the present invention provides a method for purifying FHA protein, comprising a step of removing, through a membrane chromatography process, endotoxin from the FHA protein that has been isolated in the affinity chromatography process.

Advantageous Effects of Invention In the present invention, PT and FHA proteins of Bordetella Pertussis are isolated through a purification process using an affinity column filled with a resin to which a specific compound is bound, and thus production efficiency of the PT and FHA proteins is increased. In a case where PT and FHA proteins are isolated through the purification process, the production of PT protein was remarkably increased as compared with an isolation method using an SEC process. The isolation method of PT protein or FHA protein using an affinity column decreases resin cost, process time, buffer usage, and production cost, and thus can be effectively utilized for mass production of the PT protein or FHA protein.

Brief Description of Drawings Fig. 1 illustrates a flow chart of the entire process for PT and FHA proteins. Fig. 2a illustrates a chromatogram of the isolation process for PT and FHA proteins using gradient elution in a blue affinity column process. Fig. 2b illustrates possibility of isolating PT and FHA proteins using gradient elution, as shown on SDS-PAGE. Fig. 3a illustrates a chromatogram of the isolation process for PT and FHA proteins using step elution (300 mM, 400 mM, 450 mM, 500 mM, and 1 M NaCl) in a blue affinity column process. Fig. 3b illustrates possibility of isolating PT and FHA proteins using step elution (300 mM, 400 mM, 450 mM, 500 mM, and 1 M NaCl), as shown on SDS PAGE. Fig. 4a illustrates a chromatogram of the isolation process for PT and FHA proteins using step elution (300 mM, 400 mM, 850 mM, and 1 M NaCl) in a blue affinity column process. Fig. 4b illustrates possibility of isolating PT and FHA proteins using step elution (300 mM, 400 mM, 850 mM, and 1 M NaCl), as shown on SDS-PAGE. Fig. 5a illustrates a chromatogram of the isolation process using the optimal condition for isolating PT and FHA proteins which has been established by performing a blue affinity column process with the design of experiments (DOE) in Table 1. Fig. 5b illustrates results obtained by performing the process under the optimal condition for isolating PT and FHA proteins, which has been established with the DOE in Table 1, as shown on SDS-PAGE.

Best Mode for Carrying out the Invention In an aspect of the present invention, there is provided a method for purifying PT protein or FHA protein, comprising a step of isolating, using an affinity chromatography process, PT and FHA proteins in a pre-treated Bordetella Pertussis culture. Here, the chromatography process may involve using a resin to which a compound having the structure of Formula 1 is bound:

[Formula 1]

I 0 NH

N N

C1 N N

OH The compound may be Cibacron Blue 3G-A (Sigma-Aldrich). In the present invention, the affinity columnmprocess, in which the columnmis filled with aresin to which the compound is bound, is also referred to asa "blue affinity column process" or "blue affinity chromatography process." In an embodiment of the present invention, instead of size exclusion chromatography (SE), which is aconventionally used method for isolating PT protein and FHA protein, an affinity chromatography process using the compound Cibacron Blue 3G-A as astationary phase was performed to isolate PT and FHA proteins present in aculture supernatant. In addition, the affinity chromatography process may be performed by step elution or gradient elution. Specifically, the gradient elution may be used. In an embodiment of the present invention, isolation processes for PT and FHA proteins were performed by step elution and gradient elution, respectively, to establish an effective process method for isolating PT and FHA proteins in ablue affinity chromatography process. As aresult, in acase where the process is performed by gradient elution, it was identified that the PT protein and the FHA protein are isolated well on SDS-PAGE. Here, the "gradient elution" refers to amethod of performing elution while continuously changing the mobile-phase composition. In addition, as used herein, the term "step elution" is a method of performing elution through discontinuous switching which is carried out after increased elution ability achieved by changing the concentration or composition of the solvent flowing through the column in chromatography. The elution buffer used in the gradient elution method may be a sodium phosphate solution. In addition, the elution buffer may further comprise a NaCl solution, and the blue affinity chromatography process may be performed by changing the concentration of the NaCl solution. The sodium phosphate solution may have a concentration of 10 mM to 500 mM, 20 mM to 350 mM, 50 mM to 200 mM, or 100 mM to 150 mM, and may specifically have a concentration of 100 mM. In addition, the sodium phosphate solution containing NaCl solution may have a pH of 6.5 to 8.5, 6.8 to 8.2, 7.2 to 7.8, or 7.4 to 7.7, and may specifically have a pH of 7.6. In addition, the elution buffer may be in an amount of 15 CVs to 35 CVs, 20 CVs to 33 CVs, or 23 CVs to 30 CVs, and may specifically be in an amount of 25 CVs. In addition, in the chromatography process, PT and FHA proteins may be individually eluted by gradually increasing the concentration of the NaCl solution from 0 M to 3 M, from 0 M to 2 M, or from 0 M to I M. In an embodiment of the present invention, the target proteins PT and FHA were individually eluted by flowing, in 25 CVs, 100 mM sodium phosphate solution at pH 7.6 while gradually increasing the concentration of NaCl to 1 M, in which PT was first eluted and FHA was subsequently eluted. The purification process using affinity chromatography may be subjected to a pretreatment process prior to the elution step using gradient elution. The pretreatment process may be performed through steps of: i) washing, ii) equilibrating the column using an equilibration buffer, iii) adsorbing, onto the column, a solution containing the target proteins, iv) re-equilibrating the column using a re-equilibration buffer, and v) washing the column using a washing buffer. The column filled with resin may be washed with the NaCl solution. In addition, the equilibration buffer, the re-equilibration buffer, or the washing buffer may be a sodium phosphate solution. In an embodiment of the present invention, the column was washed with 3 M NaCl solution, and the column was equilibrated and re equilibrated using a sodium phosphate solution as the equilibration buffer and the re equilibration buffer. In addition, the column was washed using a sodium phosphate solution as the washing buffer. In addition, in the present invention, the "pre-treated pertussis culture" may be obtained by steps of: 1) centrifuging a Bordetella Pertussis culture; 2) purifying the obtained supernatant through hydroxyapatite (HA) chromatography; and 3) purifying the purified supernatant through hydrophobic interaction chromatography (HIC). First, a step of centrifuging a Bordetella Pertussisculture may be performed. In the present invention, "Bordetella Pertussis", which is also called Bordetella parapertussis,is a Gram-negative coccobacillus as small as approximately 0.3 to 1 ptm. Among the major proteins of Bordetella Pertussis, PT is an abbreviation of pertussis toxin. The PT protein is produced by Bordetella Pertussis and is a toxic substance that causes dysfunction or exhibits lethal action in a living body or cell. In addition, FHA is an abbreviation of filamentous haemagglutinin adhesin, and is a virulence factor of Bordetella pertussis that causes pertussis. The PT protein and the FHA protein are outer membrane proteins that adhere to tracheal epithelial cells, are obtained from Bordetellapertussis, and are important components of pertussis vaccines. In an embodiment of the present invention, a Bordetella pertussis strain was cultured in modified stainer scholte (MSS) medium at a temperature of 35°C, and the resulting cell culture was subjected to centrifugation for 2 hours at room temperature. Then, a cell-removed culture supernatant was separated and obtained therefrom. Secondly, a step of purifying the obtained supernatant through hydroxyapatite chromatography may be performed. After centrifugation of a cell lysate or cell culture, unnecessary cell debris or the like may be removed using various column chromatography or the like. The column chromatography may be hydroxyapatite (HA) chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase-HPLC, affinity chromatography (blue affinity chromatography), or the like. In an embodiment of the present invention, hydroxyapatite chromatography was performed as a first purification process for pretreatment of a Bordetella Pertussisculture. As used herein, the term "hydroxyapatite chromatography" is column chromatography using crystalline particles of calcium phosphate as a support for separation. Negatively charged phosphate ions and positively charged calcium atoms are regularly arranged on the crystal surface. Proteins with a molecular surface that can properly have an ionic interaction with these atoms can be adsorbed onto the resin in the column. In addition, the adsorbed protein may be eluted with a phosphate buffer or the like. In an embodiment of the present invention, PT and FHA proteins were eluted using a sodium phosphate solution as an equilibrium buffer, a sodium phosphate solution as a washing buffer, and a sodium phosphate solution containing NaCl solution as an elution buffer, in hydroxyapatite chromatography. Thirdly, a step of purifying the purified supernatant through hydrophobic interaction chromatography may be performed. In an embodiment of the present invention, hydrophobic interaction chromatography was performed as a second purification process for pretreatment of a Bordetella Pertussis culture. 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 CNBr-activated agarose may be used. In addition, the hydrophobic interaction chromatography is widely used for protein isolation. In addition, in the hydrophobic interaction chromatography, for protein elution, ionic strength may be decreased or pH may be increased. In an embodiment of the present invention, a solution containing PT protein and FHA protein was eluted using a sodium phosphate solution containing NaCl solution as an equilibration buffer, a sodium phosphate solution as a washing buffer, and a sodium phosphate solution as an elution buffer, in hydrophobic interaction chromatography. In another aspect of the present invention, there is provided a method for purifying PT protein, comprising a step of removing, through a membrane chromatography (MC) process, endotoxin from the PT protein that has been isolated in the affinity chromatography process. PT and FHA, which are Bordetella Pertussis-derived proteins, contain endotoxin, a toxin present inside a bacteria. Therefore, in order to purify the PT and FHA proteins so that these proteins are utilized as vaccines, an endotoxin removal process must be carried out. The endotoxin removal may be carried out by an affinity chromatography or membrane chromatography process, and may specifically be performed by a membrane chromatography process. As used herein, the term "membrane chromatography" refers to a process used to purify monoclonal antibodies (MAbs) and other biomolecules, in which a planar macroporous membrane is used so that convection is greater than diffusion in terms of proportion, and separation efficiency of solution is relatively high. Thus, the membrane chromatography allows viruses, plasmids, macro-protein complexes, or the like to easily access the membrane and easily be isolated. Commercially available membrane chromatography may include, but is not limited to, Mustang Q (Pall Corporation), Sartobind Q (Sartorius Stedim Biotech GmbH), and the like. In an embodiment of the present invention, Mustang Q process was performed as a membrane chromatography process for removing endotoxin from PT protein. Mustang-Q is a hydrophobic polyethersulfone (PES) membrane-based capsule that removes negatively charged endotoxin by ion-exchange through a polymeric coating based on cross-linked quaternary amine groups. In the Mustang Q membrane chromatography, a pore diameter of 0.6 tm to 1.0 pm, specifically 0.7 pm to 0.9 pm, and more specifically 0.8 pm may be used. In yet another aspect of the present invention, there is provided a method for purifying FHA protein, comprising a step of removing, through a membrane chromatography process, endotoxin from the FHA protein that has been isolated in the blue affinity chromatography process. In an embodiment of the present invention, Mustang Q process was performed as a membrane chromatography process for removing endotoxin from FHA protein. In the Mustang Q membrane chromatography, a pore diameter of 0.6 pm to 1.0 pm, specifically 0.7 tm to 0.9 pm, and more specifically 0.8 tm may be used. The process for isolating and purifying PT and FHA proteins is illustrated in Fig. 1.

Mode for the Invention Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto. Example 1. Culture of Bordetella Pertussis Bordetella pertussis (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 2. Centrifugation process The cell culture was separated into the culture supernatant and the slurry by being continuously centrifuged under a condition of 9,500 rpm, with an input flow rate of 100 1/h, for 2 hours at room temperature. Example 3. HA purification Distilled water was used to remove the storage solution for hydroxyapatite (HA) resin, and then washing was performed by reaction with 1 M NaOH solution for 1 hour. Equilibration was achieved using a sodium phosphate solution as an equilibration buffer. Then, the culture supernatant obtained through the centrifugation process was adsorbed onto the HA resin, and the residual culture supernatant was removed using the equilibration buffer. Thereafter, washing was completed using a sodium phosphate solution as a washing buffer, and then PT and FHA proteins were eluted using a sodium phosphate solution containing NaCl solution as an elution buffer. Example 4. Purification on HIC column Distilled water was flowed, in 3 CVs at 120 cm/hr, through a column filled with HIC resin, to remove the storage solution. Then, 1 M NaOH solution was flowed therethrough in 4 CVs at 40 cm/hr, to perform washing. A sodium phosphate solution containing NaCl solution, as an equilibration buffer, was flowed therethrough in 5 CVs, to achieve equilibrium. After completion of the equilibration, the eluate, which had been obtained through the HA purification process, was flowed through the HIC column for adsorption. The equilibration buffer was flowed therethrough in 5 CVs, to remove the solution remaining in the column. Thereafter, a sodium phosphate solution as a washing buffer was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, a sodium phosphate solution was flowed therethrough in 10 CVs, to elute PT and FHA proteins. Example 5. Purification on blue affinity column Example 5.1. Purification by gradient elution Distilled water was flowed, in 3 CVs at 60 cm/hr, through a column filled with Cibacron Blue 3G-A resin, to remove the storage solution. Then, 3 M NaCl solution was flowed therethrough, in 3 CVs at 60 cm/hr, to perform washing before use. A sodium phosphate solution as an equilibration buffer was flowed in 5 CVs, to achieve equilibration. After completion of the equilibration, a solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at 1:1 was flowed through the column for adsorption. The equilibration buffer was flowed therethrough in 5 CVs, to remove the solution remaining in the column. Thereafter, a sodium phosphate solution as a washing buffer was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, gradient elution was performed in which a sodium phosphate solution containing NaCl solution was used as an elution buffer. Here, the NaCl solution was flowed in 10 CVs with increasing concentrations from 100 mM to 500 mM, to isolate PT protein and FHA protein. Here, the PT protein was first eluted and the FHA protein was subsequently eluted, according to difference in their binding capacity with the resin. The results are illustrated in Figs. 2a and 2b. Example 5.2. Purification by step elution Distilled water was flowed, in 3 CVs at 60 cm/hr, through a column filled with Cibacron Blue 3G-A resin, to remove the storage solution. Then, 3 M NaCl solution was flowed therethrough, in 3 CVs at 60 cm/hr, to perform washing before use. A sodium phosphate solution as an equilibration buffer was flowed in 5 CVs, to achieve equilibration. After completion of the equilibration, a solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at 1:1 was flowed through the column for adsorption. The equilibration buffer was flowed therethrough in 5 CVs, to remove the solution remaining in the column. Thereafter, a sodium phosphate solution as a washing buffer was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, step elution including 5 steps was performed. As an elution buffer for each step, a 100 mM sodium phosphate solution at pH 7.6 containing NaCl solution was used. Here, the NaCl solution was used at concentrations of 300 mM, 400 mM, 450 mM, 500 mM, and 1 M in the respective steps, and this solution was flowed in 10 CVs to isolate PT protein and FHA protein. The results are illustrated in Figs. 3a and 3b. As illustrated in Figs. 3a and 3b, it was identified that the PT protein is eluted at a NaCl concentration of 300 mM and the FHA protein is eluted starting from 400 mM, according to difference in their binding capacity. In order to identify the exact time point when the FHA protein is eluted, step elution including 4 steps was performed by varying the concentration of the NaCl solution. The NaCl solution was used at concentrations of 300 mM, 400 mM, 850 mM, and 1 M, in respective steps, and the subsequent procedure was the same as above. The results are illustrated in Figs. 4a and 4b. As illustrated in Figs. 4a and 4b, the PT protein was still eluted at a NaCl concentration of 300 mM, and the FHA protein was eluted at a NaCl concentration of 850 mM. Example 6. Establishment of optimal conditions for isolation of PT and FHA

In order to establish the optimal conditions for isolation of PT and FHA using gradient elution, a purification process was performed at a small scale by setting up the design of experiments (DOE) in Table 1.

[Table 1] C1 C2 C3 C4 C5 C6 C7 StdOrder RunOrder Center Block SP pH CV Point 1 1 3 1 1 50 7.4 20 2 2 10 1 1 150 7.4 20 3 3 9 1 1 50 7.8 20 4 4 11 1 1 150 7.8 20 5 5 1 1 1 50 7.4 30 6 6 8 1 1 150 7.4 30 7 7 7 1 1 50 7.8 30 8 8 4 1 1 150 7.8 30 9 9 5 0 1 100 7.6 25 10 10 6 0 1 100 7.6 25 11 11 2 0 1 100 7.6 25 Distilled water was flowed, in 3 CVs at 60 cm/hr, through a column filled with Cibacron Blue 3G-A resin, to remove the storage solution. Then, 3 M NaCl solution was flowed therethrough, in 3 CVs at 60 cm/hr, to perform washing before use. A sodium phosphate solution as an equilibration buffer was flowed in 5 CVs, to achieve equilibration. After completion of the equilibration, a solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at 1:1 was flowed through the column for adsorption. The equilibration buffer was flowed therethrough in 5 CVs, to remove the solution remaining in the column. Thereafter, a sodium phosphate solution as a washing buffer was flowed therethrough in 5 CVs, to perform washing. After completion of the washing, PT and FHA were isolated by performing gradient elution using, in 25 CVs, 100 mM sodium phosphate solution at pH 7.6 containing 1 M NaCl solution as an elution buffer. Here, PT was first eluted and FHA was subsequently eluted, according to difference in their binding capacity with the resin. The results are illustrated in Figs. 5a and 5b. Isolation processes were performed according to the respective conditions. As a result, PT and FHA were most effectively isolated under the conditions of 9, 10, and 11. From this, it was identified that the conditions 9, 10, and 11 are the optimal conditions for isolation of PT and FHA using gradient elution. Experimental Example 1. Identification of possibility of scale-up of established isolation conditions In order to identify whether the optimum conditions for isolation of PT and FHA, which had been established at a small scale in Example 6, are applied even in a case of scale-up, isolation processes were performed by applying the conditions at a large scale. The results are shown in Tables 2 and 3.

[Table 2] Process Batch No. PT production (mg) XaP1215 99 XaP1218 110 SEC XaP1220 100 XaP1301 112 XaP1311 106 Average production -_105

[Table 3] Process Batch No. PT production (mg) XaP1409 171 XaP1415 162 XaP1417 204 Blue Affinity XaP1418 174 XaP1604 197 XaP1605 206 Average production -_186 As shown in Tables 2 and 3, in a case where the conditions for isolation of PT and FHA, which have been established at a small scale, are applied at a large scale, PT production increased by about 80% as compared with a case where PT and FHA are isolated through an SEC process. This indicates that the conditions for isolation of PT and FHA, which have been established at a small scale, are also applicable to a large scale, and means that the blue affinity column process according to the present invention is more effective, in terms of scale-up and process yield, than the SEC process, a method for isolation of PT and FHA which has been conventionally used.

Claims (13)

1. Claims

   [Claim 1]

   A method for obtaining PT protein or FHA protein, comprising: a step of isolating, using an affinity chromatography process, PT and FHA proteins from a sample containing a Bordetella Pertussis culture.
2. [Claim 2]

   The method of claim 1, wherein the chromatography process involves using a resin to which a compound having the structure of Formula 1 is bound:

   [Formula 1]

   O NH2 O S-OH

   0 O HN I 0 -H NH

   N N CI N N H OH
3. [Claim 3]

   The method of claim 1, wherein an elution buffer for the process comprises a sodium phosphate solution.
4. [Claim 4]

   The method of claim 3, wherein the elution buffer further comprises a NaCl solution, and the process is performed by changing the concentration of the NaCl solution.
5. [Claim 5]

   The method of claim 4, wherein the concentration of the NaCl solution gradually increases from 0 M to 3 M.
6. [Claim 6]

   The method of claim 3, wherein the sodium phosphate solution has a pH of 6.5 to 8.5.
7. [Claim 7]

   The method of claim 3, wherein the sodium phosphate solution has a concentration of 50 mM to 200 mM.
8. [Claim 8]

   The method of claim 3, wherein the elution buffer is in an amount of 15 CVs to 35 CVs.
9. [Claim 9]

   The method of claim 1, wherein the following steps are further included prior to the affinity chromatography process: 1) a step of centrifuging a Bordetella Pertussisculture; 2) a step of purifying, through hydroxyapatite (HA) chromatography, the supernatant obtained in step 1); and 3) a step of purifying, through hydrophobic interaction chromatography (HIC), the supernatant purified in step 2).
10. [Claim 10]

    A method for purifying PT protein, comprising: a step of removing, through a membrane chromatography (MC) process, endotoxin from the PT protein obtained by the method of any one of claims 1 to 9.
11. [Claim 11]

    The method of claim 10, wherein in the chromatography process, a pore diameter of 0.6 tm to 1.0 pm is used.
12. [Claim 12]

    A method for purifying FHA protein, comprising: a step of removing, through a membrane chromatography (MC) process, endotoxin from the FHA protein obtained by the method of any one of claims 1 to 9.
13. [Claim 13]

    The method of claim 12, wherein in the chromatography process, a pore diameter of 0.6 pm to 1.0 pm is used.