Purification of Streptococcal Capsular Polysaccharide
OwnerPaolo Costantinoinventor
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Abstract
A purification process for the capsular polysaccharide of S. agalactiae in which the saccharide is initially treated with an aqueous mixture of an alcohol and a calcium salt, followed by precipitation with a cationic detergent. The process can be completed in less than three days and has a yield of around 60%. It avoids the need for DNase, RNase and/or protease treatment. The saccharides of the process have a very low protein contamination and a very low absorbance at 280 nm.
Claims32
A process for purifying a Streptococcus agalactiae capsular polysaccharide, comprising the steps of:
- (a) treating a suspension comprising streptococcal proteins, nucleic acids and capsular polysaccharide with an aqueous metal cation and an alcohol in order to precipitate nucleic acids and proteins;
- (b) separating the precipitated material from the aqueous material;
- and (c) treating the aqueous material with a cationic detergent in order to precipitate the capsular polysaccharide.
The process of any preceding claim, wherein the polysaccharide is a substantially full-length capsular polysaccharide.
The process of any preceding claim, wherein the polysaccharide has a molecular weight >30 kDa.
The process of any preceding claim, wherein the saccharide is partially or fully de-O-acetylated.
The process of any preceding claim, wherein the saccharide is partially or fully de-N-acetylated.
The process of any preceding claim, wherein the suspension is the supernatant from a centrifuged S. agalactiae culture.
The process of any preceding claim, wherein the alcohol is a lower alcohol.
The process of any preceding claim, wherein the alcohol is added to the suspension to give a final alcohol concentration of between 10% and 50%.
The process of any preceding claim, wherein the aqueous metal cation is monovalent or divalent.
The process of any preceding claim, wherein step (b) includes centrifugation.
The process of any preceding claim, wherein a step of diafiltration is performed after step (a) and before step (c).
The process of any preceding claim, wherein the cationic step in step (c) is a tetrabutylammonium or cetyltrimethylammonium salt, such as CTAB.
The process of any preceding claim, wherein the process further comprises re-solubilising the saccharide into aqueous medium or into alcoholic medium.
A composition comprising a Streptococcus agalactiae capsular polysaccharide, obtainable by the process of any preceding claim.
Dependencies are inferred from the claim text.