Method of separating protective components of bordetella pertussis

Abstract

To provide a method of efficiently separate protective components of Bordetella pertussis On the basis of differences in adsorbability to calcium phosphate gel formed by adding calcium ions to a Bordetella pertussis culture in the presence of excess phosphate ions, protective components of Bordetella pertussis are separated from the Bordetella pertussis culture. Traditionally, protective components of Bordetella pertussis have been separated using different purification methods for the respective components. According to the present invention, the use of the same means of purification for all subject components makes it possible to purify each component with high efficiency and high recovery rate, an aspect very advantageous for industrial production. It is also possible to efficiently produce an improved purified pertussis component vaccine comprising an effective combination of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT).

1 . A method of separating at least one member of the group consisting of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM), and pertussis toxin (PT) by bringing a Bordetella pertussis culture into contact with calcium phosphate gel which is formed by adding calcium ions to the culture in the presence of phosphate ions.  
     
     
         2 . A method of separating at least one member of the group consisting of pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP), pertussis fimbriae (FIM) and pertussis toxin (PT) by separating a Bordetella pertussis culture into cells and culture liquid, and carrying out at least one of processes (A), (B), (C) and (D): 
 (A) a process in which the separated cells are eluted with a salt solution, and pertussis filamentous hemagglutinin (FHA) is separated by bringing the eluted solution into contact with calcium phosphate gel of  claim 1 ,    (B) a process in which the cell residue resulting from the elution treatment of the above process (A) is heated in the presence of a salt solution and brought into contact with calcium phosphate gel, and pertactin (PRN, 69K-OMP) is separated by bringing the eluted solution into contact with calcium phosphate gel of  claim 1 ,    (C) a process in which the cell residue resulting from the elution treatment of the above process (A) is heated in the presence of a salt solution, the supernatant is brought into contact with calcium phosphate gel and eluted with a salt solution, and pertussis fimbriae (FIM) is separated by bringing the eluted solution into contact with calcium phosphate gel of  claim 1 ,    (D) a process in which the culture or the separated culture liquid is brought into contact with calcium phosphate gel of  claim 1 , and pertussis toxin (PT) is separated from the supernatant.    
     
     
         3 . The separation method of  claim 2 , wherein the supernatant is brought into contact with calcium phosphate gel and eluted with a salt solution to separate pertussis filamentous hemagglutinin (FHA) in process (A).  
     
     
         4 . The separation method of  claim 2 , wherein the supernatant after being brought into contact with calcium phosphate gel is brought into contact with ion exchange gel to separate pertactin (PRN, 69K-OMP) in process (B).  
     
     
         5 . The separation method of  claim 2 , wherein the supernatant is brought into contact with calcium phosphate gel and removed, and the resulting residue is eluted with a salt solution to separate pertussis fimbriae (FIM) in process (C).  
     
     
         6 . The separation method of  claim 2 , wherein the supernatant is brought into contact with ion exchange gel to separate pertussis toxin (PT) in process (D).  
     
     
         7 . The separation method of  claim 2 , wherein the salt solution used in processes (A) and (C) is a buffer containing an alkali metal salt.  
     
     
         8 . The separation method of  claim 7 , wherein the salt solution is a buffer containing 0.01-1.0 M sodium chloride.  
     
     
         9 . The separation method of  claim 1  or  2 , wherein the calcium phosphate gel is formed by adding calcium ions to the culture or the supernatant of pH 7-9 in the presence of phosphate ions.  
     
     
         10 . The separation method of  claim 9 , wherein the equivalent ratio of phosphate ions and calcium ions is 1.25-30 equivalents of phosphate ions per equivalent of calcium ions.  
     
     
         11 . The separation method of  claim 9 , wherein the calcium phosphate gel is formed by adding calcium acetate, as a calcium ion source, at 0.1-2 w/v % in the presence of a 0.05-0.1 M phosphate buffer.  
     
     
         12 . The separation method of  claim 1  or  2 , wherein at least one member of the group consisting of pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM) is separated, after which endotoxin is removed by adsorption to aluminum hydroxide gel in the presence of ammonium sulfate.  
     
     
         13 . The separation method of  claim 1  or  2 , wherein at least one member of the group consisting of pertussis toxin (PT), pertussis filamentous hemagglutinin (FHA), pertactin (PRN, 69K-OMP) and pertussis fimbriae (FIM) is separated, after which endotoxin is removed by zonal centrifugation.  
     
     
         14 . A pertussis vaccine wherein the components PT:FHA:FIM are admixed in a ratio of 4-6:8-10:1.  
     
     
         15 . A pertussis vaccine wherein the components PT:FHA:PRN:FIM are admixed in a ratio of 2-6:4-10:1-2:1.