Biotechnol. Appl. Biochem. (2003) 37, 283–287 (Printed in Great Britain) 283 Purification of capsular polysaccharide from Streptococcus pneumoniae serotype 23F by a procedure suitable for scale-up Viviane Maimoni M. Gonc  alves* 1 , Mickie Takagi*, Rodrigo B. Lima*, Hugo Massaldi †, Roberto C. Giordano‡ and Martha M. Tanizaki* *Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900, Sa  o Paulo, SP, Brazil, †Departamento de Desarrollo Biotecnolo  gico y Produccio  n, Instituto de Higiene, Montevideo, Uruguay, and ‡Dep. Engenharia Quı  mica, Universidade Federal de Sa  o Carlos, Via Washington Luiz, km 235, 13565-905, Sa  o Carlos, SP, Brazil 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 tangen- tial microfiltration through a 0.22 μm membrane, broth microfiltrate concentration by tangential ultrafil- tration in a 30 kDa spiral membrane, fractional eth- anol 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 contami- nation 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. Introduction Streptococcus pneumoniae is an encapsulated Gram-positive pathogenic bacterium that causes pneumonia, bacteraemia and meningitis. The capsule consists of a high-molecular- mass polymer whose function is to protect the micro- organism against host immune responses. Therefore, capsular polysaccharide (PS) is the main antigen used in both plain pneumococcal vaccines and those conjugated to a carrier protein [1]. The capsular PS structure is sero- type-specific and although there are 90 distinct serotypes of S. pneumoniae, most human infections are caused by only 20. A licensed 23-valent plain PS vaccine has shown pro- tective efficacy in preventing pneumococcal disease in sev- eral at-risk populations [2]. In a similar approach used to produce vaccines against pathogenic encapsulated bacteria, such as Haemophilus influenzae and Neisseria meningitidis, im- proved 7-valent or 11-valent PS protein conjugate pneumo- coccal vaccines are now undergoing clinical trial [3]. In spite of several studies carried out on these encapsulated bacteria, there is a lack of information in the literature related to large-scale processes of bacterial cultivation and capsular PS purification. The two main publications on this subject concerning S. pneumoniae are patents [4,5]. The Me  rieux protocol [4] involves the cellular lysis of the whole culture with sodium deoxycholate (DOC), the concentrationdiafiltration of the lysis product, two steps of ethanol fractionation, elimination of protein through phenol extraction, two more steps of ethanol fractionation followed by ultracentrifugation, elimination of nucleic acids by active charcoal treatment, ethanol precipitation and freeze-drying. In the Yavordios and Cousin protocol [5], the supernatant is used as the raw material and the purification method includes concentrationdiafiltration, freeze-drying, nuclease and protease treatment, chloroformbutanol ex- traction, extensive dialysis against water for 60 h, ethanol precipitation and freeze-drying. The Institut Me  rieux has also described the classical purification process of N. meningitidis serogroup A and C PS [6], which follows a method similar to the pneumococcal PS purification process and uses the detergent Cetavlon instead of DOC to cause cell lysis and to precipitate the negatively charged PS. In an improved process for meningococcal PS serogroup C purification developed in our laboratory, protein extraction by phenol was substituted by protease digestion and ultrafiltration [7]. Recently, affinity chromatography using a lectin–agarose column was described in a purification process of S. pneumoniae serotype 14 PS [8]. This method was tested for low amounts of PS (about 4 mg), and it is not clear if it could easily be scaled-up. Therefore, it would be interesting to develop new PS purification methods that eliminate the use of an organic solvent. Accordingly, this report aims to present a simple and improved method Key words : downstream processing, pneumococcal vaccine, tangential filtration. Abbreviations used : DOC, sodium deoxycholate ; K d , distribution coefficient ; PS, polysaccharide ; WHO, World Health Organization. 1 To whom correspondence should be addressed (e-mail vivimaigbutantan.gov.br).  2003 Portland Press Ltd