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 isolating a polysaccharide in a substantially pure form from bacteria selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi , the method comprising:
exposing or contacting a solution of lysed cells comprising the polysaccharide, proteins, nucleic acids, cell wall components and other impurities with silicone dioxide (SiO 2 ); and
separating the polysaccharide from the SiO 2 to isolate the polysaccharide in substantially pure form, wherein the polysaccharide is separated from the SiO 2 by filtration or by centrifugation and without using chromatography.
2. The method of claim 1 , wherein a particle size of the SiO 2 ranges from 0.01 μm to 200 μm.
3. The method of claim 1 , wherein an amount of the SiO 2 used ranges from 0.5% to 20% weight/volume (w/v).
4. The method of claim 1 , wherein the bacteria is Streptococcus pneumoniae and comprises one or more serotypes selected from the group consisting 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.
5. The method of claim 1 , wherein exposing or contacting the solution of lysed cells to the SiO 2 is carried out at a temperature ranging from 15° C. to 60° C. for a period of 10 minutes to 16 hours.
6. A method of isolating a polysaccharide in a substantially pure form from bacteria selected from the group consisting of Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenzae type b, and Salmonella typhi , the method comprising:
i) preparing a solution of lysed cells comprising the polysaccharide, proteins, nucleic acids, cell wall components and other impurities and a suspension of silicone dioxide (SiO 2 ) in water or a buffer;
ii) adding the suspension of SiO 2 to the solution of lysed cells; and
iii) isolating the polysaccharide from the SiO 2 in a substantially pure form, wherein the polysaccharide is isolated from the SiO 2 by filtration or by centrifugation and without using chromatography.
7. The method of claim 6 , wherein a size of the SiO 2 ranges from 0.01 μm to 200 μm.
8. The method of claim 6 , wherein an amount of the SiO 2 used ranges from 0.5% to 20% weight/volume (w/v).
9. The method of claim 6 , wherein the bacteria is Streptococcus pneumoniae and comprises of one or more serotypes selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11 A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F.
10. The method of claim 6 , wherein the adding step ii) is carried out at a temperature ranging from 15° C. to 60° C. for a period of 10 minutes to 16 hours.
11. The method of claim 2 , wherein the size of the SiO 2 ranges from 3 μm to 40 nm.
12. The method of claim 7 , wherein the size of the SiO 2 ranges from 3 μm to 40 μm.