Production method for capsular polysaccharide having pneumococcal serotype

1. A method of producing a capsular polysaccharide having a pneumococcal serotype, the method comprising:

(a) cultivating bacterial cells that produce a pneumococcal serotype while maintaining pH of a culture broth in the range of 7.0 to 9.4;

(b) terminating the cultivating of step (a) at a time between when the absorbance of the culture broth remains constant and when the absorbance begins to decrease;

(c) performing additional cultivating of the culture broth of step (b) without addition of a pH adjuster until the pH of the culture broth reaches pH of 5.5 or lower;

(d) adding a lysing agent to the culture broth obtained from step (c) to lyse cells, precipitating proteins, and removing the precipitated proteins and cell debris to obtain a clarified cell lysate; and

(e) isolating and purifying the capsular polysaccharide from the lysate obtained from step (d).

2. The method of claim 1 , wherein the pneuomococcal serotype is 1, 2, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 22F, 23F or 33F.

3. The method of claim 1 , wherein the cultivating of step (a) is performed at 34-38° C. under agitation at 50-150 rpm.

4. The method of claim 1 , wherein step (b) is performed by terminating the cultivating of step (a) within 1 to 3 hours from the time when the absorbance of the culture broth remains constant.

5. The method of claim 1 , wherein the additional cultivating of step (c) is performed at 34-38° C. under agitation at 50-150 rpm without pH adjustment.

6. The method of claim 1 , wherein the lysing agent used in step (d) is sodium deoxycholate.

7. The method of claim 1 , wherein step (d) is performed by adding the lysing agent to the culture broth obtained from step (c) to lyse cells, then incubating the resulting cell lysate at 10-20° C. for 3-24 hours without agitation to precipitate proteins, and removing the precipitated proteins and cell debris by centrifugation.

8. The method of claim 1 , wherein the isolating and purifying of step (e) comprises:

(i) filtering the lysate obtained from step (d) using a depth filter;

(ii) concentrating a filtrate obtained from step (i), followed by ultrafiltration and centrifugation;

(iii) reacting a supernatant obtained from step (ii) with a cationic surfactant, and then centrifuging the resulting solution to obtain a pellet or supernatant containing capsular polysaccharides;

(iv) reacting the capsular polysaccharides obtained from step (iii) with sodium iodide, followed by centrifugation, thereby obtaining a supernatant;

(v) adding activated carbon to the solution obtained from step (iv), followed by filtration; and

(vi) concentrating a filtrate obtained from step (v), followed by ultrafiltration and centrifugation, thereby obtaining capsular polysaccharides.

9. The method of claim 8 , wherein the concentrating of step (ii) is performed using a 100 kDa membrane.

10. The method of claim 8 , wherein the concentrating of step (iv) is performed using a 30 kDa membrane.

11. The method of claim 8 , wherein the cationic surfactant used in step (iii) is cetyltrimethylammonium bromide.

12. The method of claim 11 , wherein the cetyltrimethylammonium bromide is used at a concentration of 0.5˜3.0%.

13. The method of claim 8 , wherein the activated carbon used in step (v) used at a concentration of 1-5% (w/v).