Production of high yields of bacterial polysaccharides

1. A method for preparing a capsular polysaccharide of Neisseria meningitidis X with high yield and high purity, comprising the steps of:

(a) preparing an aqueous fermenter nutrient medium comprising casamino acid;

(b) inoculating the fermenter nutrient medium with a Neisseria meningitidis X bacterium having optical density at 590 nm of 0.8 to 1.2;

(c) continuous exponential feeding with a feed solution with a feed addition rate varying between 10 mL/hr/1.5 L and 60 mL/hr/1.5 L beginning at an OD at 590 nm between 3 and 4;

(d) incubating the inoculated fermenter nutrient medium under controlled conditions of pH, temperature and dissolved oxygen percentage;

(e) harvesting the capsular polysaccharide produced in step (d) when the optical density (OD) at 590 nm is less than 60% of the highest culture OD at 590 nm;

(f) purifying the capsular saccharide obtained in step (e);

(g) optionally concentrating the purified capsular polysaccharide obtained in step (f); and wherein said purified polysaccharide has yield from 300 to 550 mg/L, average molecular weight from 400 to 550 KDa, purification step recovery from 60% to 65% and contains less than 0.5% proteins/peptides, less than 0.5% nucleic acids and less than 5 EU/μg endotoxins.

2. A process for the production of Neisseria meningitides capsular polysaccharide as claimed in claim 1 wherein:

(a) said capsular polysaccharide producing bacteria is selected from N. meningitidis X strains M8210, M9601, M9591, M9592, M9554, M2526, 247X and 5967(ST 750);

(b) said fermenter nutrient medium comprises:

(i) casamino acids at an amount 5 g/L to 20 g/L, 0.14 g/L to 0.19 g/L Ammonium chloride, 10 g/L to 11 g/L dextrose, 5.8 g/L to 6 g/L Sodium chloride, 0.9 g/L to 1 g/L Potassium sulphate, 3.8 g/L to 4 g/L Potassium phosphate dibasic, 4.8 g/L to 5 g/L Glutamic acid, 0.2 g/L to 0.3 g/L L-Arginine, 0.4 g/L to 0.5 g/L L-Serine, 0.24 g/L to 0.25 g/L L-Cysteine, 0.18 g/L to 0.19 g/L Magnesium chloride, 0.02 g/L Calcium chloride and 0.002 g/L Ferrous sulphate such that concentration of components of the medium may vary by ±10%; or

(ii) casamino acids at an amount 5 g/L to 10 g/L, 0.14 g/L to 0.17 g/L Ammonium chloride, 10 g/L to 11 g/L dextrose, 5.8 g/L to 6 g/L Sodium chloride, 0.9 g/L to 1 g/L Potassium sulphate, 3.8 g/L to 4 g/L Potassium phosphate dibasic, 4.8 g/L to 5 g/L Glutamic acid, 0.2 g/L to 0.3 g/L L-Arginine, 0.4 g/L to 0.5 g/L L-Serine, 0.24 g/L to 0.25 g/L L-Cysteine, 0.18 g/L to 0.19 g/L Magnesium chloride, 0.02 g/L Calcium chloride and 0.002 g/L Ferrous sulphate such that concentration of components of the medium may vary by ±10%;

(c) said inoculation of fermenter nutrient medium is by a seed inoculum having an optical density of 0.8 to 1;

(d) said continuous exponential feeding begins with initial feed addition at a rate of 10 mL/hr/1.5 L when OD at 590 nm is between 3 to 4 which gradually increases to 30 mL/hr/1.5 L until the culture OD at 590 nm is highest and then maintained at 60 mL/hr/1.5 L until culture OD at 590 nm reaches 50% of highest OD at 590 nm when culture can be harvested;

(e) said feed solution comprises of 72 to 76 g/L dextrose, 38 g/L to 42 g/L Sodium glutamate, 2.8 g/L to 3.2 g/L L-Arginine, 2.8 g/L to 3.2 g/L L-Serine, 1.9 g/L to 2.1 g/L L-Cysteine, 1.9 g/L to 2 g/L Magnesium chloride, 0.13 g/L to 0.15 g/L Calcium chloride and 0.02 g/L Ferrous sulphate such that concentration of components of the feed solution may vary by ±10%;

(f) said incubation is carried out

(i) at a temperature of 36° C. to 37° C., at a pH of 7-7.2, at 350 to 500 rpm, dissolved oxygen percentage of 15% to 25%, Gas flow at 1 to 1.5, Air at 0 to 100% and Oxygen at 0 to 100% for 7 to 10 hours or

(ii) at a temperature of 36° C., at a pH 7.2, at about 400 rpm, dissolved oxygen percentage of 25%, Gas flow at 1 to 1.5;

(iii) Air at 0 to 100% and Oxygen at 0 to 100% for 8 to 9 hours;

(g) said inactivation is carried out using 1% formaldehyde for 1 to 2 hrs at 37° C. followed by incubation at 10° C. for 30 minutes;

(i) said harvesting is carried out by

(i) centrifugation at 14500 g for 45 minutes to remove all cell residues to obtain a supernatant;

(ii) subjecting the supernatant to 0.2μ filtration followed by 100 KD diafiltration and concentration with water for injection (WFI);

and

(h) said process is carried out in continuous fed-batch culture system.

3. The process of claim 1 wherein step (f) comprises:

(a) removal of protein and endotoxin impurities by utilizing anionic detergent at a concentration from 1 to 1.2% in combination with a chelating agent at a concentration from 1.5 to 3 mM and alcohol at a concentration from 40% to 50%;

(b) addition of sodium acetate at a concentration from 4 to 6% for removal of nucleic acids;

(c) addition of a cationic detergent at 3 to 4% for binding polysaccharides and impurities;

(d) precipitation of polysaccharides from cationic detergent-polysaccharide complex by utilizing sodium chloride at 0.05 M in presence of 96% alcohol;

(e) removal of protein and nucleic acid impurities by washing the precipitated polysaccharides with 45% alcohol in the presence of sodium chloride at a concentration of 0.4 M;

(f) selective precipitation of polysaccharides by utilizing 96% alcohol;

(g) dissolving the precipitated polysaccharides in WFI and subjecting the dissolved polysaccharides to tangential flow filtration;

and wherein said purification process does not utilize any chromatography.

4. The process of claim 3 , wherein purification comprises the following steps:

(a) removal of protein and endotoxin impurities by utilizing deoxycholate at a concentration of 1% in combination with ethylenediaminetetraacetic acid at a concentration of 2 mM and ethanol at a concentration of 40%;

(b) addition of 4 to 6% sodium acetate for removal of nucleic acids;

(c) addition of cetyltrimethylammonium bromide at a concentration from 3 to 4% for binding polysaccharides and impurities;

(d) precipitation of polysaccharides from the cetyltrimethylammonium bromide-polysaccharide complex by utilizing sodium chloride at a concentration of 0.05 M in presence of 96% ethanol;

(e) removal of protein and nucleic acid impurities by washing the precipitated polysaccharides with ethanol at a concentration of 45% in the presence of sodium chloride at a concentration of 0.4 M;

(f) selective precipitation of polysaccharides by utilizing 96% ethanol;

(g) dissolving the precipitated polysaccharides in WFI and subjecting the dissolved polysaccharides to tangential flow filtration;

and wherein said purification process does not utilize any chromatography.

5. The method according to claim 1 , further comprising step (h) wherein purified N. meningitidis X polysaccharide is sized to an average size of between 100 and 150 Kda by using high pressure cell disruption system.

6. The method according to claim 5 , further comprising the step of conjugating said size reduced polysaccharide to a carrier protein, to yield a protein-polysaccharide conjugate and formulating an immunogenic composition comprising said protein-polysaccharide conjugate.

7. The method according to claim 6 , wherein said N.meningitidis X polysaccharide is conjugated to a carrier protein selected from the group consisting of Tetanus toxoid (TT), Diphtheria toxoid (DT), Cross Reaching Material (Mutant Diphtheria toxoid) (CRM197), fragment C of TT, protein D, Outer Membrane Protein Complex (OMPC) and pneumolysin.

8. The method according to claim 6 , wherein said N. meningitidis X polysaccharide is conjugated to the carrier protein via a hetero or homo-bifunctional linker by cyanylation.

9. The method according to claim 8 , wherein said cyanylation reagent is selected from a group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), or 2-cyanopyridazine-3(2H) one (2-CPO), or a functional derivative or modification thereof.

10. An immunogenic composition prepared according to claim 6 , wherein further the protein-polysaccharide conjugates are in a lyophilized form.

11. An immunogenic composition prepared according to claim 6 , wherein further the protein-polysaccharide conjugates are in a buffered liquid form.

12. An immunogenic composition prepared according to claim 6 , wherein further one or more N.meningitidis protein-polysaccharide conjugates are optionally adsorbed onto aluminium hydroxide, aluminium phosphate or a mixture of both or unadsorbed onto an adjuvant.

13. An immunogenic composition according to claim 12 , further comprising an aluminium salt adjuvant at an amount of 25-125 μg of Al +++ per 0.5 mL.

14. An immunogenic composition according to claim 13 , further comprising a preservative selected from thiomersal and 2-phenoxyethanol.

15. An immunogenic lyophilized monovalent or multivalent vaccine composition containing a N. meningitidis serogroup X polysaccharide-protein conjugate prepared according to claim 10 wherein said vaccine is given as a 1, 5 or 10 dose formulation.

16. A method for preparing a N. meningitidis serogroup X capsular polysaccharide with high yields and high purity which comprises the steps of:

(i) preparing a fermentation medium composition containing casamino acids and ammonium chloride and inoculating the composition with a Neisseria meningitides serogroup X bacterium composition having an optical density at 590 nm of 0.8 to 1.2;

(ii) continuous exponential feeding with a feed solution with a feed addition rate varying between 10 mL/hr/1.5 L and 60 mL/hr/1.5 L beginning at an OD at 590 nm between 3 and 4;

(iii) incubating the inoculated fermenter nutrient medium under controlled conditions of pH, temperature and dissolved oxygen percentage;

(iv) harvesting the capsular polysaccharide produced in step (iii) when the optical density (OD) at 590 nm is less than 60% of the highest culture OD at 590 nm; and

(v) recovering and purifying the Neisseria meningitidis serogroup X capsular polysaccharide obtained in (iv);

and wherein said purified Neisseria meningitidis serogroup X polysaccharide yields from 300 to 550 mg/L, has an average molecular weight from 400 to 550 KDa, contains less than 0.5% proteins/peptides, less than 0.5% nucleic acids, less than 5 EU/μg endotoxins and said purification step recovery is from 60% to 65%.

17. A method for preparing a Neisseria meningitides serogroup X capsular polysaccharide with high yields and high purity which comprises the steps of:

i) preparing a fermentation medium composition comprising a combination of casamino acid and at least one additional nitrogen source selected from soya peptone and yeast extract and inoculating the composition with a Neisseria meningitidis X bacterium composition having an optical density at 590 nm of 0.8 to 1.2;

(ii) continuous exponential feeding with a feed solution with a feed addition rate varying between 10 mL/hr/1.5 L and 60 mL/hr/1.5 L beginning at an OD at 590 nm between 3 and 4 at 590 nm;

(iii) incubating the inoculated fermenter nutrient medium under controlled conditions of pH, temperature and dissolved oxygen percentage;

(iv) harvesting the capsular polysaccharide produced in step (iii) when the optical density (OD) at 590 nm is less than 60% of the highest culture OD at 590 nm; and

(v) recovering and purifying the Neisseria meningitidis X capsular polysaccharide obtained in (iv) without using chromatography;

and wherein said purification purified Neisseria meningitidis X polysaccharide has yields from 300 to 550 mg/L, has an average molecular weight from 400 to 550 KDa, contains less than 0.5% proteins/peptides, less than 0.5% nucleic acids, less than 5 EU/μg endotoxins and said purification step recovery is from 60% to 65%.

18. A method for preparing a Neisseria meningitides serogroup X capsular polysaccharide with high yields and high purity which comprises the steps of:

i) providing a fermentation medium composition comprising a combination of casamino acid, ammonium chloride and at least one additional nitrogen source selected from soya peptone and yeast extract and inoculating the composition with a Neisseria meningitidis X bacterium composition having an optical density at 590 nm of 0.8 to 1.2;

(ii) continuous exponential feeding with a feed solution with a feed addition rate varying between 10 mL/hr/1.5 L and 60 mL/hr/1.5 L beginning at an OD at 590 nm between 3 and 4;

(iii) incubating the inoculated fermenter nutrient medium under controlled conditions of pH, temperature and dissolved oxygen percentage;

(iv) harvesting the capsular polysaccharide produced in step (iii) when the optical density (OD) at 590 nm is less than 60% of the highest culture OD at 590 nm; and

(v) recovering and purifying the Neisseria meningitidis X capsular polysaccharide obtained in (iv) without using chromatography;

wherein said purification purified Neisseria meningitidis X polysaccharide yields from 300 to 550 mg/L, has an average molecular weight from 400 to 550 KDa, contains less than 0.5% proteins/peptides, less than 0.5% nucleic acids, less than 5 EU/μg endotoxins and said purification step recovery is from 60% to 65%.