Purification of capsular polysaccharide from Streptococcus pneumoniae serotype 23F by a procedure suitable for scale‐up

Abstract

Streptococcus pneumoniae is a pathogenic encapsulated bacterium, which causes pneumonia, bacteraemia and meningitis. Capsular polysaccharide conjugated to a carrier protein has been widely used as a vaccine antigen. Serotype 23F is one of the prevalent worldwide pneumococci. A simple and efficient method for capsular polysaccharide serotype 23F purification that can easily be scaled-up was developed. This method consisted of using culture broth obtained by tangential microfiltration through a 0.22 microm membrane, broth microfiltrate concentration by tangential ultrafiltration in a 30 kDa spiral membrane, fractional ethanol precipitation (28-60%), nuclease and proteinase treatment, and concentration/diafiltration in a 30 kDa cassette membrane. The final polysaccharide recovery was 89%. The final protein and nucleotide contamination was 1.5% (w/w) and 0.3% (w/w) respectively. The final pure polysaccharide meets the requirements of the World Health Organization and residual proteinase was not found in the final product.

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

“…Residual proteolytic activity of trypsin, pronase and nagarse was determinated in the Wnal product [13].…”

“…Our laboratory has developed an improved method for puriWcation of vaccines polysaccharides against Neisseria meningitidis serotype C [12] and Streptococcus pneumoniae serotype 23 and 6B [13,14]. This process reduces considerably the number of ethanol precipitation steps, phenol extraction was replaced by enzymatic treatment and the ultracentrifugation by ultraWltration in the presence of chelating agent and detergent.…”

Production of native flagellin from Salmonella Typhimurium in a bioreactor and purification by tangential ultrafiltration

“…Our group has successfully applied tangential ultrafiltration, with different cut-off sizes, to the purification of various biomolecules derived from microorganisms (Gonçalves et al, 2003;Takagi, et al, 2008;Oliveira et al, 2010). In the present report we describe conditions that allow production of Salmonella FliCi flagellin in bioreactors with subsequent purification in high yields by tangential ultrafiltrafiltration.…”

DEVELOPMENT OF A NEW PROCESS FOR PURIFICATION OF CAPSULAR POLYSACCHARIDE FROM Streptococcus pneumoniae SEROTYPE 14

“…Tanizaki et al (1996) eliminated the use of phenol, replacing it by enzymatic treatment and ultrafiltration for the Neisseria meningitides serogroup C capsular PS. This process also proved to be efficient for capsular polysaccharide purification of other microorganisms (Gonçalves et al, 2003;Gonçalves et al, 2007;Takagi et al, 2008). However, after the enzymatic treatment, it is necessary to remove the hydrolyzed material and the enzymes, which may also cause loss of the product of interest.…”

“…In our laboratory, purification processes were developed for purification of capsular polysaccharides of Neisseria meningitidis (Tanizaki et al, 1996), Streptococcus pneumoniae (Gonçalves et al, 2003;Gonçalves et al, 2007) and Haemophilus influenzae type b (Takagi et al, 2008). In these processes, the number of ethanol precipitation steps and centrifugations were reduced and the use of phenol was completely eliminated, being replaced by treatment with endonucleases and proteases, followed by tangential ultrafiltration, wherein the low molecular weight hydrolysate material was removed while PS was retained in the concentrate.…”