Method for separation of protein and other impurities from microbial capsular polysaccharides
The invention relates to a method for the removal of protein and other impurities from microbial capsular polysaccharides. More particularly, the present invention relates to isolation of microbial capsular polysaccharides in pure form after removal of protein and other impurities.
1. A method for preparing an immunogenic composition comprising a purified capsular polysaccharide conjugated to a carrier protein selected from the group consisting of diphtheria toxoid, tetanus toxoid, and CRM 197 , the method comprising:
exposing or contacting a solution of lysed Streptococcus pneumoniae cells comprising the capsular polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO 2 );
separating the capsular polysaccharide from the SiO 2 by filtration or by centrifugation and without using chromatography to isolate the capsular polysaccharide in substantially pure form; and
conjugating the purified capsular polysaccharide to the carrier protein.
2. The method of claim 1 , wherein the Streptococcus pneumoniae comprises one or more serotypes selected from the group comprising of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F.
3. The method of claim 1 , wherein the SiO 2 comprises particles each having a size ranging from 0.01 μm to 200 μm.
4. The method of claim 1 , wherein the solution of lysed cells is exposed or contacted with the SiO 2 at a temperature ranging from 15° C. to 60° C. for a period of time ranging from 10 minutes to 16 hours.
5. The method of claim 1 , wherein the SiO 2 is in an amount ranging from 0.5% to 20% weight/volume (w/v).