Purification of staphylococcus aureus type 5 and type 8 capsular saccharides

Abstract

The invention provides a method for releasing capsular polysaccharide from S. aureus type 5 or type 8 cells, comprising the step of treating the cells with acid. The invention further provides a process for purifying capsular polysaccharide from S. aureus type 5 or type 8 cells comprising this method. Other processing steps may be included in the process, such as enzymatic treatment, e.g. to remove nucleic acid, protein and/or peptidoglycan contaminants; diafiltration, e.g. to remove low molecular weight contaminants; anion exchange chromatography, e.g. to remove residual protein; and concentration.

We claim: 
     
         1 : A method for releasing capsular polysaccharide from  S. aureus  type 5 or 8 cells, comprising the step of treating the cells with acid. 
     
     
         2 : The method of  claim 1 , wherein the cells are in the form of a wet cell paste or are suspended in an aqueous medium. 
     
     
         3 : The method of  claim 1 , wherein the acid treatment is carried out using acetic acid. 
     
     
         4 : The method of  claim 1 , wherein the acid treatment results in the capsular polysaccharide having a degree of O-acetylation between 60-100%. 
     
     
         5 : The method of  claim 1 , wherein the method further comprises a step of neutralisation. 
     
     
         6 : The method of  claim 1 , wherein the method further comprises a step of centrifugation of the cells and collection of the polysaccharide-containing supernatant. 
     
     
         7 : A process for purifying capsular polysaccharide from  S. aureus  type 8 cells comprising the method of  claim 1 . 
     
     
         8 : The process of  claim 7 , wherein the process further comprises a step of treatment of the capsular polysaccharide with DNase and/or RNase. 
     
     
         9 : The process of  claim 7 , wherein the process further comprises a step of treatment of the capsular polysaccharide with mutanolysin. 
     
     
         10 : The process of  claim 7 , wherein the process further comprises a step of diafiltration. 
     
     
         11 : The process of  claim 10 , wherein the diafiltration is tangential flow filtration. 
     
     
         12 : The process of  claim 7 , wherein the process farther comprises a step of anion exchange chromatography. 
     
     
         13 : The process of  claim 7 , wherein the process further comprises a step of gel filtration. 
     
     
         14 : The process of  claim 7 , wherein the process further comprises a step of concentration of the polysaccharide. 
     
     
         15 : The process of  claim 7 , wherein the molecular mass of the purified polysaccharide is between 2-3500 kDa. 
     
     
         16 : The process of  claim 7 , wherein the process further comprises a step of depolymerisation of the purified polysaccharide to form an oligosaccharide. 
     
     
         17 : The process of  claim 7 , wherein the process further comprises a step of sterile filtration. 
     
     
         18 : The process of  claim 7 , wherein the process provides a composition comprising the polysaccharide and a level of peptidoglycan contamination that is less than 5% by weight peptidoglycan relative to the total weight of the polysaccharide. 
     
     
         19 : The process of  claim 18 , wherein the level of peptidoglycan contamination is about 2%. 
     
     
         20 : The process of  claim 7 , wherein the process provides a composition comprising the polysaccharide and a level of protein contamination that is less than 5% by weight protein relative to the total weight of the polysaccharide. 
     
     
         21 : The process of  claim 7 , wherein the process provides a composition comprising the polysaccharide and a level of nucleic acid contamination that is less than 1% by weight nucleic acid relative to the total weight of the polysaccharide. 
     
     
         22 : The process of  claim 7 , wherein the process further comprises a step of conjugation to a carrier molecule. 
     
     
         23 : A composition comprising an  S. aureus  type 5 or type 8 capsular polysaccharide obtainable by the process of  claim 7  or a conjugate obtainable by the process of claim 
     
     
         24 : The composition of  claim 23  further comprising one or more  S. aureus  protein antigen(s) selected from the group consisting of a clfA antigen; a clfB antigen; a sdrE2 antigen; a sdrC antigen; a sasF antigen; a emp antigen; a sdrD antigen; a spa antigen; a esaC antigen; a esxA antigen; a esxB antigen; a sta006 antigen; a isdC antigen; a Hla antigen; a sta011 antigen; a isdA antigen; a isdB antigen; and a sta073 antigen. 
     
     
         25 : The composition of  claim 24 , wherein the one or more  S. aureus  protein antigen(s) are selected from the group consisting of a esxA antigen; a esxB antigen; a sta006 antigen; a Hla antigen; a sta011 antigen; and a sta073 antigen. 
     
     
         26 : The composition of  claim 25 , wherein the composition comprises S,  aureus  protein antigens according to one of combinations (1) to (10) below:
 (1) a esxA antigen, a esxB antigen, a sta006 antigen and a Hla antigen;   (2) a esxA antigen, a esxB antigen, a sta006 antigen and a sta011 antigen;   (3) a esxA antigen, a esxB antigen and a sta011 antigen;   (4) a esxA antigen, a esxB antigen, a Hla antigen, a sta006 antigen and a sta011 antigen;   (5) a esxA antigen, a esxB antigen and a Hla antigen;   (6) a Hla antigen, a sta006 antigen and a sta011 antigen;   (7) a esxA antigen and a esxB antigen;   (8) a esxA antigen, a esxB antigen and a sta006 antigen;   (9) a esxA antigen, a esxB antigen, a sta011 antigen and a sta073 antigen; and a sta006 antigen and a sta011 antigen.