JP7031814B2 - Methods for Obtaining Bordetella Pertussis-Derived Proteins, Including Affinity Chromatography Steps - Google Patents

Description

The present invention relates to a method for obtaining a protein derived from Bordetella pertussis, which comprises an affinity chromatography step.

Whooping cough is an acute respiratory illness that occurs primarily in infants and is characterized by a cough that lasts for more than 2 weeks. Bordetella pertussis, a gram-negative and aerobic bacillus, has been reported to cause whooping cough. Bordetella pertussis uses humans as its sole host and infects humans primarily through the respiratory tract. In addition, Bordetella pertussis inhabits the mucous membranes of the respiratory tract and causes disease in the human body. In the 1930s, a cellular pertussis vaccine was developed and proved to have a protective effect against whooping cough. In addition, the pertussis vaccine was used in combination with the tetanus and diphtheria inactivated killed vaccine in the 1940s, but side effects (attacks, edema, fever, etc.) of the whole-cell pertussis vaccine have been reported. Therefore, it has been required to develop a safe pertussis vaccine.

In the 1950s, studies on the etiology of Bordetella pertussis were conducted, and components such as pertussis toxin (PT), fibrous hemagglutinin (FHA), pertactin (PRN), and fimbria (FIM) were reported as antigens. .. Since then, the development of a cell-free pertussis vaccine containing the isolation and purification of these proteins has been promoted. Since the 1980s, the purified pertussis vaccine was first developed and vaccinated in Japan.

The purpose of purifying the pertussis vaccine is to produce endotoxin-free products that are rich in certain proteins such as PT, FHA and PRN. Conventionally, the antigen has been purified at the same time by repeating ammonium sulfate precipitation and density gradient centrifugation. However, such a method has a drawback that a large amount 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 Patent Application Publication No. 2015-0124973 discloses an acellular pertussis vaccine composition comprising PT, FHA and FIM types 2 and 3.

Existing methods for purifying PT and FHA, the major proteins of Bordetella pertussis, are those present in culture supernatants using size exclusion chromatography (SEC), a method used in the preparation of common vaccines. And to isolate FHA. However, this method is difficult to scale and the process yield is low. Furthermore, in a method for producing a pertussis vaccine that involves mixing three or four components, even a decrease in the productivity of one component not only has the problem of prolonging the overall production period, but also the production batch. There may be financial problems caused by the increase in numbers.

In this regard, we have combined specific compounds during studies to find ways to solve the problems associated with the SEC process and enable efficient production of the pertussis-derived proteins PT and FHA. It has been found that when PT and FHA proteins are isolated by affinity chromatography using existing resins, not only is the time and cost required for the method significantly reduced, but the production of target proteins is increased. .. Based on this finding, the inventors have completed the invention.

Therefore, an object of the present invention is to provide a method for isolating a PT protein or an FHA protein, which is a protein derived from Bordetella pertussis, using an affinity column.

Another object of the present invention is to provide a method for purifying a PT protein or an FHA protein which is a protein derived from Bordetella pertussis.

To achieve the above object, the present invention provides a method for isolating a PT protein or FHA protein in a pretreated Bordetella pertussis culture by an affinity chromatography step.

To achieve the above object, the present invention provides a method for purifying a PT protein, which comprises a step of removing endotoxin from the PT protein isolated in the affinity chromatography step by a membrane chromatography (MC) step. ..

Further, the present invention provides a method for purifying an FHA protein, which comprises a step of removing endotoxin from the FHA protein isolated in the affinity chromatography step by a membrane chromatography step.

In the present invention, the PT and FHA proteins of Bordetella pertussis are isolated by a purification step using an affinity column filled with a resin to which a specific compound is bound, so that the production efficiency of the PT and FHA proteins is increased. When the PT and FHA proteins were isolated by the purification step, the production of PT protein was significantly increased as compared with the isolation method using the SEC step. The method of isolating PT protein or FHA protein using an affinity column can be efficiently used for mass production of PT protein or FHA protein in order to reduce resin cost, process time, buffer usage and production cost. ..

The flow chart of the whole method for PT and FHA proteins is shown. The chromatogram of the isolation process of PT and FHA proteins using gradient elution in the blue affinity column process is shown. Shows the possibility of isolation of PT and FHA proteins using gradient elution, shown by SDS-PAGE. The chromatogram of the PT and FHA protein isolation step using step elution (300 mM, 400 mM, 450 mM, 500 mM and 1 M NaCl) in the blue affinity column step is shown. It shows the possibility of isolation of PT and FHA proteins using stepwise elution (300 mM, 400 mM, 450 mM, 500 mM and 1 M NaCl) shown by SDS-PAGE. The chromatogram of the PT and FHA protein isolation step using step elution (300 mM, 400 mM, 850 mM and 1 M NaCl) in the blue affinity column step is shown. It shows the possibility of isolation of PT and FHA proteins using stepwise elution (300 mM, 400 mM, 850 mM and 1 M NaCl) shown by SDS-PAGE. The chromatogram of the isolation step using optimal conditions for isolation of PT and FHA proteins, established by performing the blue affinity column step in the design of experiments (DOE) in Table 1, is shown. The results obtained by performing the method under the conditions optimal for the isolation of the PT and FHA proteins established by DOE in Table 1 shown by SDS-PAGE are shown.

One aspect of the invention provides a method of purifying a PT protein or FHA protein, comprising the step of isolating the PT and FHA proteins in a pretreated Bordetella pertussis culture using an affinity chromatography step. To.
As used herein, the chromatographic step is Formula 1.

の構造を有する化合物が結合している樹脂を使用することを含み得る。

It may include using a resin to which a compound having the above structure is bound.

The compound may be Cibacron Blue 3G-A (Sigma-Aldrich). In the present invention, the affinity column step in which the column is filled with the resin to which the compound is bound is also referred to as a "blue affinity column step" or a "blue affinity chromatography step".

In one embodiment of the invention, affinity using the compound Cibacron Blue 3G-A as the stationary phase instead of size exclusion chromatography (SEC), a method conventionally used for isolation of PT and FHA proteins. A chromatography step was performed to isolate the PT and FHA proteins present in the culture supernatant.

In addition, the affinity chromatography step can be performed by step elution or gradient elution. In particular, gradient elution can be used. In one embodiment of the invention, an efficient treatment method for isolating PT and FHA proteins in a blue affinity chromatography step by performing the PT and FHA protein isolation steps by stepwise elution and gradient elution, respectively. Was established. As a result, it was confirmed that PT protein and FHA protein were successfully isolated by SDS-PAGE when the treatment was performed by gradient elution.

As used herein, "gradient elution" refers to a method of elution while continuously changing the composition of the mobile phase. Moreover, as used herein, the term "step elution" is by intermittent switching made after increasing the elution capacity by varying the concentration or composition of the solvent flowing through the column in chromatography. This is a method of elution.

The elution buffer used in the gradient elution method may be a sodium phosphate solution. In addition, the elution buffer may further contain a NaCl solution and the blue affinity chromatography step may be performed by varying 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 have a concentration of 100 mM in particular. Further, the sodium phosphate solution containing the 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. It may have a pH of 7.6 in particular. Further, the elution buffer may be in an amount of 15 CV to 35 CV, 20 CV to 33 CV or 23 CV to 30 CV, and in particular, an amount of 25 CV. Further, in the chromatography step, the PT and FHA proteins can be eluted individually by gradually increasing the concentration of the NaCl solution to 0M to 3M, 0M to 2M, or 0M to 1M.

In one embodiment of the invention, the target proteins PT and FHA are individually eluted by flowing a 100 mM sodium phosphate solution (pH 7.6) at 25 CV while gradually increasing the concentration of NaCl to 1 M. , PT was eluted first, followed by FHA.

Purification steps using affinity chromatography can be subjected to pretreatment steps prior to elution steps using gradient elution. The pretreatment steps are i) a step of washing, ii) a step of equilibrating the column using an equilibration buffer, iii) a step of adsorbing a solution containing a target protein to the column, and iv) a re-equilibration buffer. This can be done by a step of rebalancing the column using v) and a step of washing the column using a wash buffer.

Columns filled with resin can be washed with NaCl solution. Further, the equilibration buffer, the re-equilibration buffer, or the washing buffer may be a sodium phosphate solution. In one embodiment of the invention, the column was washed with 3M NaCl solution and the column was equilibrated and re-equilibrated using sodium phosphate solution as equilibration buffer and re-equilibration buffer. In addition, the column was washed with sodium phosphate solution as wash buffer.

Further, in the present invention, the "pretreated whooping cough culture" includes 1) a step of centrifuging the pertussis culture and 2) a step of purifying the obtained supernatant by hydroxyapatite (HA) chromatography. 3) The purified supernatant can be obtained by the step of purifying by hydrophobic interaction chromatography (HIC).

First, a step of centrifuging the B. pertussis culture can be performed.

In the present invention, "Bordetella pertussis" is also called Bordetella parapertussis and is a gram-negative bacillus having a size of about 0.3 μm to 1 μm. Among the major proteins of Bordetella pertussis, PT is an abbreviation for pertussis toxin. PT protein is a toxic substance produced by Bordetella pertussis that causes or is lethal in vivo or in cells. Furthermore, FHA is an abbreviation for fibrous hemagglutinin adhesin, which is a virulence factor of Bordetella pertussis that causes whooping cough. PT protein and FHA protein are outer membrane proteins that attach to tracheal epithelial cells, are obtained from Bordetella pertussis, and are important components of the pertussis vaccine.

In one embodiment of the invention, whooping cough strains were cultured in modified Steiner scholte (MSS) medium at a temperature of 35 ° C. and the resulting cell cultures were centrifuged at room temperature for 2 hours. The culture supernatant from which the cells had been removed was then separated and obtained.

Second, the resulting supernatant can be purified by hydroxyapatite chromatography.

After centrifugation of the cell lysate or cell culture, various column chromatography and the like can be used to remove unwanted cell debris and the like. Column chromatography includes hydroxyapatite (HA) chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel exclusion chromatography, gel filtration chromatography, HPLC, reverse phase HPLC, affinity chromatography (blue affinity chromatography). Etc. may be used. In one embodiment of the invention, hydroxyapatite chromatography was performed as a first purification step for pretreatment of Bordetella pertussis culture.

As used herein, the term "hydroxyapatite chromatography" is column chromatography using crystalline particles of calcium phosphate as a carrier for separation. Negatively charged phosphate ions and positively charged calcium atoms are regularly arranged on the crystal surface. Proteins with molecular surfaces capable of appropriately having ionic interactions with these atoms can be adsorbed on the resin in the column. Further, the adsorbed protein can be eluted with a phosphate buffer or the like.

In one embodiment of the invention, in hydroxyapatite chromatography, a sodium phosphate solution is used as an equilibrium buffer, a sodium phosphate solution is used as a wash buffer, and a sodium phosphate solution containing a NaCl solution is used as an elution buffer. It was used to elute PT and FHA proteins.

Third, the purified supernatant can be purified by hydrophobic interaction chromatography.

In one embodiment of the invention, hydrophobic interaction chromatography was performed as a second purification step for pretreatment of B. pertussis cultures.

As used herein, the term "hydrophobic interaction chromatography" refers to a separation method that uses a hydrophobic interaction between a matrix having hydrophobic functional groups and a molecule. The matrix can have a hydrophobic function by denaturation of the hydrophilic and inert agarose. Modified agarose obtained by reacting an alkylamine with CNBr-activated agarose may be used. In addition, hydrophobic interaction chromatography is widely used for protein isolation. In addition, in hydrophobic interaction chromatography, ionic strength may be reduced or pH may be increased due to protein elution.

In one embodiment of the invention, in hydrophobic interaction chromatography, a sodium phosphate solution containing a NaCl solution is used as an equilibrium buffer, a sodium phosphate solution is used as a wash buffer, and sodium phosphate is used as an elution buffer. The solution was used to elute the solution containing PT and FHA proteins.

In another aspect of the invention, there is provided a method of purifying a PT protein, comprising removing endotoxin from the PT protein isolated in the affinity chromatography step by a membrane chromatography (MC) step.

Bordetella pertussis-derived proteins PT and FHA contain endotoxin, a toxin present in the bacterium. Therefore, in order to purify PT and FHA proteins and utilize these proteins as vaccines, endotoxin removal steps must be performed.

The endotoxin removal may be carried out by an affinity chromatography step or a membrane chromatography step, and in particular, it can be carried out by a membrane chromatography step.

As used herein, the term "membrane chromatography" is a monoclonal in which a macroporous flat membrane is used so that convection is proportionately greater than diffusion and solution separation efficiency is relatively high. Refers to the steps used to purify antibodies (MAbs) and other biomolecules. Therefore, membrane chromatography allows viruses, plasmids, macroprotein complexes, etc. to easily reach the membrane and be easily isolated. Commercially available membrane chromatographies may include, but are not limited to, Mustang Q (Pall Corporation), Sartorius Q (Sartorius Stedim Biotech GmbH) and the like.

In one embodiment of the invention, the Mustang Q step was performed as a membrane chromatography step for removing endotoxin from the PT protein. Mustang-Q is a hydrophobic polyether sulfone (PES) membrane-based capsule that removes negatively charged endotoxins by ion exchange via a polymer coating based on crosslinked quaternary amine groups. In Mustang Q membrane chromatography, pore diameters of 0.6 μm to 1.0 μm, particularly 0.7 μm to 0.9 μm, and even more particularly 0.8 μm can be used.

In yet another aspect of the invention, there is provided a method of purifying an FHA protein, comprising removing endotoxin from the FHA protein isolated in the blue affinity chromatography step by a membrane chromatography step.

In one embodiment of the invention, the Mustang Q step was performed as a membrane chromatography step for removing endotoxin from the FHA protein.

In Mustang Q membrane chromatography, pore diameters of 0.6 μm to 1.0 μm, particularly 0.7 μm to 0.9 μm, and even more particularly 0.8 μm may be used.

The steps of isolating and purifying the PT and FHA proteins are shown in FIG.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples merely exemplify the present invention and do not limit the scope of the present invention to the examples.

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

Example 2. Centrifugation Step The cell culture was separated into culture supernatant and slurry by continuous centrifugation at room temperature for 2 hours at an inflow rate of 100 l / h under the condition of 9500 rpm.

Example 3. After removing the preservative solution for hydroxyapatite (HA) resin using HA purified distilled water, washing was performed for 1 hour by reaction with 1M NaOH solution. Equilibration was performed using a sodium phosphate solution as the equilibration buffer. Next, the culture supernatant obtained by the centrifugation step was adsorbed on the HA resin, and the remaining culture supernatant was removed using an equilibration buffer. After that, washing was completed using sodium phosphate solution as wash buffer, and then PT and FHA proteins were eluted using sodium phosphate solution containing NaCl solution as elution buffer.

Example 4. Purification on HIC Column Distilled water was flowed through a column filled with HIC resin at 120 cm / hour at 3 CV to remove the preservative solution. Next, a 1 M NaOH solution was flowed through the column at 4 CV at 40 cm / hour for washing. A sodium phosphate solution containing a NaCl solution as an equilibration buffer was flowed through a column at 5 CV for equilibration. After the equilibration was completed, the eluate obtained by the HA purification step was poured onto a HIC column and adsorbed. The equilibration buffer was run through the column at 5 CV to remove any residual solution in the column. Then, a sodium phosphate solution as a washing buffer was flowed through the column at 5 CV for washing. After washing was complete, a sodium phosphate solution was run through the column at 10 CV to elute the PT and FHA proteins.

Example 5. Purification Example with Blue Affinity Column 5.1. Purification by gradient elution Distilled water was flowed at 3 CV at 60 cm / hour on a column filled with Cibacron Blue 3GA resin to remove the preservative solution. Next, a 3M NaCl solution was flushed through the column at 3 CV at 60 cm / hour for washing prior to use. Equilibration was performed by flowing a sodium phosphate solution as an equilibration buffer at 5 CV. After the equilibration was completed, the solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at a ratio of 1: 1 was poured into a column and adsorbed. The equilibration buffer was run through the column at 5 CV to remove any residual solution in the column. After that, a sodium phosphate solution as a washing buffer was flowed through the column at 5 CV for washing.

After the washing was completed, gradient elution was performed using a sodium phosphate solution containing a NaCl solution as an elution buffer. Here, the NaCl solution was flowed at 10 CV at an increased concentration of 100 mM to 500 mM to isolate the PT protein and the FHA protein. Here, due to the difference in binding ability with the resin, the PT protein was eluted first, and then the FHA protein was eluted. The results are shown in FIGS. 2a and 2b.

Example 5.2. Purification by step elution Distilled water was flowed at 3 CV at 60 cm / hour on a column filled with Cibacron Blue 3GA resin to remove the preservative solution. Next, a 3M NaCl solution was flushed through the column at 3 CV at 60 cm / hour for washing prior to use. Equilibration was performed by flowing a sodium phosphate solution as an equilibration buffer at 5 CV. After the equilibration was completed, the solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at a ratio of 1: 1 was poured into a column and adsorbed. The equilibration buffer was run through the column at 5 CV to remove any residual solution in the column. Then, a sodium phosphate solution as a washing buffer was flowed through the column at 5 CV for washing.

After the washing was completed, step elution including 5 steps was performed. A 100 mM sodium phosphate solution (pH 7.6) containing a NaCl solution was used as the elution buffer for each step. In this case, NaCl solutions were used at concentrations of 300 mM, 400 mM, 450 mM, 500 mM and 1 M at each step and the solution was run at 10 CV to isolate PT and FHA proteins. The results are shown in FIGS. 3a and 3b. As shown in FIGS. 3a and 3b, it was confirmed that the PT protein was eluted at a NaCl concentration of 300 mM and the FHA protein was eluted starting from 400 mM due to the difference in binding ability.

In order to identify the exact time point at which the FHA protein was eluted, step elution was performed, including 4 steps, by varying the concentration of the NaCl solution. NaCl solutions were used at concentrations of 300 mM, 400 mM, 850 mM and 1 M at each step and the subsequent procedure was the same as above. The results are shown in FIGS. 4a and 4b. As shown 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. Establishing Optimal Conditions for Isolation of PT and FHA In order to establish optimal conditions for isolation of PT and FHA using gradient elution, the purification step was reduced by establishing the design of experiments (DOE) in Table 1. I went on a scale.

Distilled water was flowed at 3 CV at 60 cm / hour on a column filled with Cibacron Blue 3GA resin to remove the preservative solution. Next, a 3M NaCl solution was flushed through the column at 3 CV at 60 cm / hour for washing prior to use. Equilibration was performed by flowing a sodium phosphate solution as an equilibration buffer at 5 CV. After the equilibration was completed, the solution obtained by mixing the HIC eluate of Example 1.4 and distilled water at a ratio of 1: 1 was poured into a column and adsorbed. The equilibration buffer was run through the column at 5 CV to remove any residual solution in the column. Then, a sodium phosphate solution as a washing buffer was flowed through the column at 5 CV for washing.

After completion of washing, PT and FHA were isolated by performing gradient elution using 100 mM sodium phosphate solution (pH 7.6) containing 1 M NaCl solution as elution buffer at 25 CV. In this case, due to the difference in binding ability with the resin, PT was eluted first, and then FHA was eluted. The results are shown in FIGS. 5a and 5b.

The isolation step was performed according to the respective conditions. As a result, PT and FHA were most efficiently isolated under the conditions of 9, 10 and 11. From this, it was confirmed that the conditions 9, 10 and 11 are the optimum conditions for the isolation of PT and FHA using gradient elution.

Experimental example 1. Confirmation of the possibility of scale-up of established isolation conditions To confirm whether the optimum conditions for isolation of PT and FHA established on a small scale in Example 6 apply even when the scale is expanded. , The isolation step was carried out by applying these conditions on a large scale. The results are shown in Tables 2 and 3.

As shown in Tables 2 and 3, when the conditions for isolation of PT and FHA established on a small scale are applied on a large scale, PT production is such that PT and FHA are isolated by the SEC process. It increased by about 80% compared to the case of. This indicates that the conditions for isolation of PT and FHA established on a small scale are applicable to a large scale, and the blue affinity column process according to the present invention has been conventionally used for PT and FHA. It means that it is more effective in terms of scale expansion and process yield than the SEC process which is the isolation method of.

Claims (9)

A method for obtaining PT protein or FHA protein, which comprises the step of isolating PT (pertussis toxin) protein and FHA (fibrous hemagglutinin) protein from a sample containing a pertussis culture using an affinity chromatography step. And,
The elution buffer for the affinity chromatography step comprises a sodium phosphate solution.
The elution buffer further comprises a NaCl solution and
The concentration of the NaCl solution gradually increased from 0M to 3M,
The affinity chromatography step is the formula 1.

A method comprising the use of a resin to which a compound having the structure of is bound . The method of claim 1 , wherein the sodium phosphate solution has a pH of 6.5-8.5. The method of claim 1 , wherein the sodium phosphate solution has a concentration of 50 mM to 200 mM. The method of claim 1 , wherein the elution buffer is in an amount of 15 CV to 35 CV. Prior to the affinity chromatography step, the following steps:
1) The step of centrifuging the B. pertussis culture and
2) The step of purifying the supernatant obtained in step 1) by hydroxyapatite (HA) chromatography, and
3) The method of claim 1, further comprising purifying the supernatant purified in step 2) by hydrophobic interaction chromatography (HIC). A method for purifying a PT protein, which comprises a step of removing endotoxin from the PT protein obtained by the method according to any one of claims 1 to 5 by a membrane chromatography (MC) step . The method according to claim 6 , wherein a pore size of 0.6 μm to 1.0 μm is used in the membrane chromatography step. A method for purifying an FHA protein, which comprises a step of removing endotoxin from the FHA protein obtained by the method according to any one of claims 1 to 5 by a membrane chromatography (MC) step. The method according to claim 8 , wherein a pore size of 0.6 μm to 1.0 μm is used in the membrane chromatography step.

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