Process for detergent-free production of outer membrane vesicles of a gram-negative bacterium

1. A process for producing a detergent-free preparation of bacterial outer membrane vesicles (OMV) for use in vaccines, said process comprising the steps of:

(a) cultivating a population of Gram-negative bacteria in culture to stationary growth phase;

(b) beginning at a time at least about 1 hour after onset of the stationary growth phase, incubating the bacteria obtained in step (a) in a medium adjusted to, or having a pH higher than, pH 8.0 and a concentration of a metal chelating agent of between about 1 and 100 mM to extract said bacterial OMV; and

(c) after step (b), recovering the extracted bacterial OMV by separation of the bacterial OMV from the bacteria.

2. The process according to claim 1 , wherein the Gram-negative bacteria comprise a genetic modification which causes the bacteria to produce a lipopolysaccharide (LPS) with reduced toxicity compared to a corresponding wild-type LPS while retaining at least part of the LPS adjuvant activity, which genetic modification:

(i) decreases or knocks-out expression of one or more of the following genes:

(A) lpxL1 or a gene the sequence of which is at least 95% identical with SEQ ID NO:1;

(B) lpxL2; and

(C) lpxK;

and/or

(ii) effects expression of one or more of genes lpxE and/or pagL.

3. The process according to claim 1 , wherein the time after onset of the stationary growth phase during which the bacteria are incubated in the medium in step (b) is between about 1 and 9 hours.

4. The process according to claim 1 , further comprising a step of sterilizing the bacterial OMV.

5. The process according to claim 1 , wherein the concentration of the metal chelating agent in step (b) is between about 5 mM and 15 mM, and/or wherein the pH is between:

(i) pH 8.0 and pH 9.5, or

(ii) pH 8.0 and pH 9.0, or

(iii) pH 8.2 and pH 8.8, or

(iv) pH 8.4 and pH 8.7.

6. The process according to claim 1 , wherein the cultivating of step (a) and/or the incubating of step (b) is performed in a medium volume of at least about 10 liters.

7. The process according to claim 1 , wherein the Gram-negative bacteria are a species of the genera Neisseria or Bordetella.

8. The process according to claim 1 wherein the Gram-negative bacteria comprise one or more mutations that decrease or knock-out expression of a gene product.

9. The process according to claim 1 , wherein the Gram-negative bacteria are of multiple PorA subtypes.

10. The process according to claim 1 , wherein the bacterial population comprises Gram-negative bacteria of more than one strain wherein bacteria of each strain are of different PorA subtypes.

11. The process according to claim 1 , wherein the Gram-negative bacteria express an antigen that is foreign to the strain or species of said bacteria.

12. The process according to claim 1 , further comprising a step of combining the recovered bacterial OMV with a pharmaceutically acceptable excipient.

13. The process according to claim 1 wherein the chelating agent is EDTA.

14. The process according to claim 3 wherein the time after onset of the stationary growth phase during which the bacteria are incubated in the medium in step (b) is between about 2 and 5 hours.

15. The process according to claim 7 wherein the bacterial species is Neisseria meningitidis or Bordetella pertussis.

16. The process according to claim 8 wherein the gene product whose expression is decreased or knocked out is Cps or a product active in lipid A biosynthesis selected from the group consisting of LpxL1, RmpM, PorA, PorB and OpA.