Methods and compositions for increasing toxin production

1. A method for obtaining one or more Clostridium difficile ( C. difficilel ) toxins comprising the steps of:

(a) preparing an aqueous growth medium comprising soy peptone and D-sorbitol;

(b) inoculating the medium with a C. difficile bacterium;

(c) culturing the inoculated medium under conditions which facilitate growth of bacterium and toxin production; and

(d) isolating the one or more C. difficile toxins from the growth medium.

2. The method of claim 1 , wherein:

(a) in step (a) the growth medium comprises: (i) between 10 and 20 g/L soy peptone; (ii) between 10 and 30 g/I yeast extract; between 2 and 5 g/L NaHCO 3 ; between 1 and 10 g/L sodium phosphate, dibasic; between 1 and 10 g/L potassium phosphate, monobasic; and between 6 and 20 g/L D-sorbitol;

(b) in step (c) the inoculated medium is at pH of between 6.35 and 6.65 during culturing;

(c) in step (c) the culturing of the inoculated medium takes place at 37° C. to 41° C.;

(d) the culturing of the inoculated media is carried out under anaerobic conditions; or

(e) in step (b) the medium is inoculated with an aqueous C. difficile culture.

3. The method of claim 1 , further comprising the step of detoxifying the isolated one or more C. difficile toxins to prepare one or more toxoids.

4. The method of claim 3 , wherein:

(a) steps (b) and (c) are repeated more than once, with inoculation into fresh growth medium in each repeat;

(b) wherein step (c) takes place at 37° C. to 41° C.;

(c) step (d) comprises: removing from the growth medium viable C. difficile organisms and spores, separating the one or more toxins from the growth media, and purifying the one or more toxins; or

(d) step (e) comprises reacting the one or more toxins with the addition of formaldehyde.

5. The method of claim 1 , wherein:

(a) the growth medium further comprises:

at least one additive selected from the group consisting of chromium trioxide, clindamycin, ascorbic acid, butyric acid, D(+)xylose, sucrose, and a combination of azaserine, adenosine, and biotin.

6. The method of claim 5 , wherein the growth medium comprises:

(a) at least two of said additives;

(b) D-sorbitol at a concentration between 6 g/L and 20 g/L;

(c) D-sorbitol at a concentration of 12 g/L;

(d) chromium trioxide at a concentration between 40 and 60 mg/L;

(e) chromium trioxide at a concentration of 50 mg/L;

(f) clindamycin at a concentration between 0.4 and 0.6 mg/L;

(g) clindamycin at a concentration of 0.5 mg/L;

(h) ascorbic acid at a concentration between 2.5 g/L and 10 g/L;

(i) ascorbic acid at a concentration selected from 2.5 g/L and 10 g/L;

(j) butyric acid at a concentration between 30 mM and 60 mM;

(k) butyric acid at a concentration selected from 30 mM and 60 mM;

(I) D(+)xylose at a concentration between 6 and 10 g/L;

(m) D(+)xylose at a concentration of 6 g/L;

(n) adenosine at a concentration of between 0.8 and 1.2 mM, biotin at a concentration of between 40 and 60 nM, and azaserine at a concentration between 15 and 50 μM; or

(o) adenosine at a concentration of 1 mM, biotin at a concentration of 50 nM, and

azaserine at a concentration of 50 μM.

7. The method of claim 6 , wherein the growth medium comprises soy peptone, yeast extract, KH 2 PO 4 , Na 2 HPO 4 , and NaHCO 3 , and wherein the culture is at a pH of between 6.35 and 7.45.

8. A method of enhancing the production from a C. difficile culture of Toxin B relative to the production of Toxin A comprising the steps of:

(a) preparing an aqueous growth medium comprising soy peptone and D-sorbitol;

(b) inoculating the medium with a C. difficile bacterium; and

(c) culturing the inoculated medium at 37° C. to 41° C.

9. The method of claim 8 , wherein:

(a) step (c) takes place at 37° C. and at a pH of pH 6.5;

(b) the pH of step (c) is controlled at a pH between pH 6.35 to pH 6.65; or

(c) the aqueous growth medium of step (a) further comprises between 10 and 20 g/L soy peptone; and between 6 and 20 g/L D-sorbitol.

10. The method of claim 8 , wherein:

(a) the production of Toxin A relative to Toxin B is less than 3:1;

(b) the production of Toxin A relative to Toxin B is less than 2:1; or

(c) the production of Toxin A relative to Toxin B is equal to or less than 1.5:1.