METHOD FOR OBTAINING PURIFIED BACTERIAL POLYSACCHARIDES

Abstract

The present disclosure relates to a method for obtaining purified bacterial polysaccharides. The method comprises simultaneous removal of impurities as well as sizing of bacterial polysaccharides using an acid instead of conventional mechanical sizing methods. The method is simple, rapid and cost effective. The method results in high polysaccharide recovery and low impurity content. The purified polysaccharide obtained by the method of the present disclosure may be used for large scale production of polysaccharide-protein conjugate vaccines.

Claims

1. A method for obtaining purified and sized bacterial polysaccharides, the method comprising the following steps: (a) providing a fermentation harvest comprising bacterial cell, polysaccharide, proteins, nucleic acid and cell debris; and (b) treating the fermentation harvest with an acid to separate the polysaccharide from protein, nucleic acid and cell debris to obtain purified and sized bacterial polysaccharides.

2. The method as claimed in claim 1, wherein the purified and sized bacterial polysaccharides have a protein content less than 3%, a nucleic acid content less than 2%, and a molecular size in a range of 50 kDa to 600 kDa (SEC-HPLC), wherein the recovery of polysaccharides is at least 60%.

3. The method as claimed in claim 1, wherein the purified and sized bacterial polysaccharides has a polysaccharide polydispersity index/coefficient less than 2 and a CWPs content not more than 2 mol %.

4. The method as claimed in claim 1, wherein the bacterial polysaccharides are derived from a bacteria selected from the group comprising Streptococcus spp, Salmonella spp., Shigella spp., E. coli, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus pneumonia, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Enterococcus faecalis, Enterococcus faecium, Bacillus anthracis, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Clostridium spp., Mycobacterium spp., Moraxella catarrhalis, Klebsiella pneumoniae, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Ehrlichia spp., and Rickettsia spp.

5. The method as claimed in claim 4, wherein the bacterial polysaccharides are derived from a Streptococcus spp. selected from the group comprising Group A Streptococcus, Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus viridans.

6. The method as claimed in claim 5, wherein the bacterial polysaccharides are derived from Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48.

7. The method as claimed in claim 4, wherein the bacterial polysaccharides are derived from a Salmonella spp. selected from the group comprising Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, and Salmonella typhimurium.

8. The method as claimed in claim 4, wherein the bacterial polysaccharides are derived from Neisseria meningitidis serotype selected from the group consisting of A, B, C, E29, H, I, K, L, M, W135, X, Y, and Z.

9. The method as claimed in claim 4, wherein the bacterial polysaccharides are derived from a Staphylococcus spp. selected from the group comprising Staphylococcus aureus, Staphylococcus aureus type 5, and Staphylococcus aureus type 8.

10. The method as claimed in claim 1, wherein the pH of the fermentation harvest is in the range of 5.8 to 6.8.

11. The method as claimed in claim 1, wherein the acid is selected from the group consisting of trifluoroacetic acid, trichloroacetic acid, sulphuric acid, phosphoric acid, acetic acid, hydrochloric acid, nitric acid.

12. The method as claimed in claim 1, wherein the concentration of the acid in the fermentation harvest is in the range of 0.1 M to 20 M.

13. The method as claimed in claim 1, wherein the concentration of the acid is in the range of 1 M to 10 M.

14. The method as claimed in claim 1, wherein the concentration of the acid is in the range of 1 M to 4M.

15. The method as claimed in claim 1, wherein the concentration of the acid is in the range of 2 M to 5 M.

16. The method as claimed in claim 1, wherein the pH during step (b) is less than 2.

17. The method as claimed in claim 1, wherein the pH during step (b) is less than 1.5.

18. The method as claimed in claim 1, wherein the pH during step (b) is less than 1.0.

19. The method as claimed in claim 1, wherein the pH during step (b) is less than 0.5.

20. The method as claimed in claim 1, wherein the temperature during step (b) is in the range of 2 C. to 100 C.

21. The method as claimed in claim 1, wherein the temperature during step (b) is in the range of 10 C. to 90 C.

22. The method as claimed in claim 1, wherein the temperature during step (b) is in the range of 20 C. to 80 C.

23. The method as claimed in claim 1, wherein the temperature during step (b) is in the range of 20 C. to 60 C.

24. The method as claimed in claim 1, wherein the molecular size of purified and sized bacterial polysaccharides is in the range of 10 kDa to 1000 kDa.

25. The method as claimed in claim 1, wherein the molecular size of purified and sized bacterial polysaccharides is in the range of 50 kDa to 300 kDa.

26. The method as claimed in claim 1, wherein the fermentation harvest is treated with the acid for a time period in the range of 1 hour to 10 hours.

27. The method for producing purified and sized bacterial polysaccharides as claimed in claim 1, further comprising the following steps: (a) providing a fermentation harvest comprising bacterial cell; (b) subjecting the fermentation harvest to centrifugation to separate cell free supernatant; (c) concentrating and filtering the cell free supernatant of step (b); (d) treating the concentrated and filtered cell free supernatant of step (c) with an acid having concentration in the range of 2 M to 5M at 20 C. to 60 C. and pH in the range of 0.1 to 2; (e) subjecting the acid treated mixture of step (d) to centrifugation to remove precipitated impurities; (f) incubating the supernatant of step (e) at a temperature in the range of 20 C. to 50 C. for a time period in the range of 4 hours to 48 hours; (g) raising the pH of the solution of step (f) using a pH adjusting agent; (h) diafiltering the solution of step (g) to increase the concentration of polysaccharide in the solution; and (i) filtering the concentrated polysaccharide of step (h) to obtain purified and sized polysaccharide having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, polydispersity less than 2, molecular size in the range of 10 kDa to 600 kDa (SEC-HPLC) and >10% methyl pentose content.

28. The method as claimed in claim 27, wherein the filtration in step (c) is carried out on 100 kDa tangential flow filtration (TFF) membrane at a pH in the range of 5.5 to 8 and the diafiltration in step (h) is carried out on 10 kDa tangential flow filtration (TFF) membrane at a pH in the range of 4 to 6.

29. The method as claimed in claim 27, wherein the filtration in step (i) is carried out using 0.2 filter at a pH in the range of 6 to 7.

30. The method as claimed in claim 1, wherein the fermentation harvest is treated with a lytic agent prior to treatment with an acid.

31. The method as claimed in claim 30, wherein the lytic agent is selected from mechanical and non-mechanical lytic agents.

32. The method as claimed in claim 31, wherein the mechanical lytic agent is selected from sonication, high pressure homogenization and bead mill.

33. The method as claimed in claim 31, wherein the non-mechanical lytic agent is a chemical lytic agent selected from acids, alkali and detergents.

34. The method as claimed in claim 33, wherein the chemical lytic agent is selected from the group consisting of deoxycholate sodium (DOC), sodium dodecyl sulphate (SDS), triton X, tween (20, 80), EDTA, urea, cetyltrimethylammonium bromide (CTAB), decanesulfonic acid, tert-octylphenoxy poly(oxy ethylene)ethanols, octylphenol ethylene oxide condensates, N-lauryl sarcosine sodium (NLS), lauryl iminodipropionate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate, cholate.

35. The method as claimed in claim 31, wherein the non-mechanical lytic agent is a physical lytic agent selected from thermal lysis, cavitation and osmotic shock.

36. The method as claimed in claim 31, wherein the wherein the non-mechanical lytic agent is an enzyme selected from lysozyme, lysostaphin, zymolase, proteinase K, protease and glycanase.

37. The method for producing purified and sized bacterial polysaccharides as claimed in claim 1, further comprising the following steps: (a) providing a fermentation harvest comprising bacterial cell; (b) treating the fermentation harvest with a lytic agent to obtain a mixture of polysaccharide, proteins, nucleic acid and cell debris; (c) subjecting the lytic agent treated fermentation harvest of step (b) to centrifugation to separate cell free supernatant; (d) concentrating and filtering the cell free supernatant of step (c); (e) treating the concentrated and filtered cell free supernatant of step (d) with an acid having concentration in the range of 2 M to 5M at 20 C. to 60 C. and pH in the range of 0.1 to 2; (f) subjecting the acid treated mixture of step (e) to centrifugation to remove precipitated impurities; (g) incubating the supernatant of step (f) at a temperature in the range of 20 C. to 50 C. for a time period in the range of 4 hours to 48 hours; (h) raising the pH of the solution of step (g) using a pH adjusting agent; (i) diafiltering the solution of step (h) to increase the concentration of polysaccharide in the solution; and (j) filtering the concentrated polysaccharide of step (i) to obtain purified and sized polysaccharide having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, polydispersity less than 2, molecular size in the range of 10 kDa to 600 kDa (SEC-HPLC) and >10% methyl pentose content.

38. The method as claimed in claim 37, wherein step (b) is carried out at a pH in the range of 5 to 7.

39. The method as claimed in claim 37, wherein the filtration in step (d) is carried out on 100 kDa tangential flow filtration (TFF) membrane at a pH in the range of 5.5 to 8 and the diafiltration in step (i) is carried out on 10 kDa tangential flow filtration (TFF) membrane at a pH in the range of 4 to 6.

40. The method as claimed in claim 37, wherein the filtration in step (j) is carried out using 0.2 filter.

41. The method as claimed in claim 1, wherein the recovery of the polysaccharides is at least 60%.

42. The method as claimed in claim 1, wherein a pH adjusting agent is used to raise the pH in the range of 5.5 to 6.5.

43. The method as claimed in claim 42, wherein the pH adjusting agent is selected from disodium hydrogen phosphate and dipotassium hydrogen phosphate.

44. The method as claimed in claim 1, further comprising at least one chromatography step to obtain the purified and sized polysaccharides, wherein the at least one chromatography step is selected from the group consisting of ion-exchange (cationic or anionic), affinity chromatography, hydrophilic-interaction, hydrophobic-interaction, size-exclusion, hydroxyl apatite, gel-permeation chromatography, Cibacron Blue pseudo affinity sorbent, mixed mode chromatography sorbent, membrane chromatography, Capto Adhere, TOYOPEARL MX-Trp-650M, Cellufine MAX AminoButyl, monolith chromatography device, adsorbent chromatography, lectin agarose column, and Amberlite column.

45. The method as claimed in claim 1, further comprising treatment with one or more chemical/biological reagents at any stage of the method, the one or more chemical/biological reagents selected from the group consisting of cetyl trimethyl ammonium bromide (CTAB), hexadimethrine bromide and myristyltrimethylammonium, triton, acetate, sodium carbonate, zinc, enzymes, alcohol (Ethanol, Isopropanol), phenol, acetone, salts (magnesium, calcium), sodium dodecyl sulfate (SDS), polysorbates, sodium sarcosine, NaCl, urea, formaldehyde, ammonium chloride, ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, mineral acid, organic acid, metal cations, toluene, chloroform, alkali, ascorbic acid, tetrabutylammonium, potassium chloride, alkyl sulfates, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium -olefin sulfonates, sodium alkyl sulfonates, -sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and sodium alkyl glyceryl ether sulfonates, DNase and/or RNase, Benzonase, Mutanolysin/lysozyme, beta.-D-N-acetyl glucosaminidase, and Proteinase K.

46. The method as claimed in claim 1, wherein the purified and sized polysaccharides are subjected to an additional size reduction step selected from thermal treatment, sonic treatment, chemical hydrolysis, endolytic enzyme treatment, and physical shear, Gaulin-homogenizer, and sonication.

47. A bacterial polysaccharide obtained by the method as claimed in claim 1, having a protein content less than 3%, a nucleic acid content less than 2%, a molecular size in the range of 50 kDa to 600 kDa (SEC-HPLC), and a polysaccharide recovery of at least 60%.

48. The bacterial polysaccharide as claimed in claim 47, wherein the molecular size is in the range of 100 kDa to 300 kDa (SEC-HPLC).

49. The bacterial polysaccharide as claimed in claim 47, wherein the polysaccharide is used for preparation of plain polysaccharide.

50. The bacterial polysaccharide as claimed in claim 47, the polysaccharide is used for preparation of polysaccharide-protein conjugate vaccine.

51. An immunogenic composition comprising Streptococcus pneumoniae polysaccharides having a protein content less than 3%, a nucleic acid content less than 2%, a CWPs content not more than 2 mol %, and a molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC), wherein the polysaccharides are derived from Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48.

52. The immunogenic composition as claimed in claim 51, wherein the polysaccharides are conjugated to carrier protein selected from CRM197, tetanus toxoid (TT), diphtheria toxoid (DT), Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD), cholera toxin B, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins or any equivalents thereof.

53. The immunogenic composition as claimed in claim 51, wherein the polysaccharides are conjugated to the carrier protein using one of cyanylation chemistry, CNBr chemistry, reductive amination chemistry, and carbodiimide chemistry.

54. The immunogenic composition as claimed in claim 52, wherein the cyanylation agent is selected from 1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP), 1-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 2-cyanopyridazine-3(2H)one (2-CPO), and a functional derivative or modification thereof.

55. The immunogenic composition as claimed in claim 52, wherein the polysaccharides are conjugated to the carrier protein in the presence of a linker selected from the group comprising hexanediamine, ethylene diamine, hydrazine, adipic dihydrazide, and 1,6-diaminooxyhexane.

56. The immunogenic composition as claimed in claim 52, wherein the polysaccharides are conjugated to the carrier protein in the absence of a linker.

57. The immunogenic composition as claimed in claim 51, wherein serotype 2 has a methyl pentose content of at least 50%, serotype 18C has a methyl pentose content of at least 25% and serotype 22F has a methyl pentose content of at least 35%.

58. The immunogenic composition as claimed in claim 51, further comprising pharmaceutically acceptable excipients selected from surfactants, stabilizers, buffers, diluents, adjuvants, preservatives and solvents.

59. The immunogenic composition as claimed in claim 58, wherein the preservative is selected from the group comprising 2-phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, methyl paraben, propyl paraben, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, benzyl alcohol and combinations thereof.

60. The immunogenic composition as claimed in claim 51, wherein the composition is one of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30-valent pneumococcal-protein-conjugate composition.

61. An immunogenic composition comprising 10 distinct Streptococcus pneumoniae polysaccharide-protein conjugates having a protein content less than 3%, a nucleic acid content less than 2%, CWPs content not more than 2 mol %, and molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC), and wherein the polysaccharides are derived from serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F, and all the polysaccharides are conjugated to CRM197.

62. An immunogenic composition comprising 17 distinct Streptococcus pneumoniae polysaccharide-protein conjugates having a protein content less than 3%, a nucleic acid content less than 2%, CWPs content not more than 2 mol %, and molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC), wherein the polysaccharides are derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F; wherein serotype 3 is conjugated to CRM197, serotype 18C is conjugated to CRM197, serotype 4 is conjugated to DT, serotype 15B is conjugated to TT and serotype 22F is conjugated to TT.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present disclosure will now be described with the help of the accompanying drawing, in which

[0052] FIG. 1 illustrates the literature .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 22F;

[0053] FIG. 2 illustrates the .sup.1H NMR spectrum purified S. pneumoniae polysaccharide for serotype 22F obtained by the method of present disclosure;

[0054] FIG. 3 illustrates the literature spectrum of purified S. pneumoniae polysaccharide for serotype 18C;

[0055] FIG. 4 illustrates the reference .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 18C;

[0056] FIG. 5 illustrates the .sup.1H NMR spectrum purified S. pneumoniae polysaccharide for serotype 18C obtained by the method of present disclosure;

[0057] FIG. 6a illustrates the literature .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 2;

[0058] FIG. 6b illustrates the chemical structure of S. pneumoniae polysaccharide for serotype 2; and

[0059] FIG. 7 illustrates the .sup.1H NMR spectrum purified S. pneumoniae polysaccharide for serotype 2 obtained by the method of present disclosure.

DETAILED DESCRIPTION

[0060] Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.

[0061] Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.

[0062] The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms a, an, and the may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms comprises, comprising, including, and having, are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.

[0063] The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.

[0064] The present disclosure envisages a simple and cost effective method for obtaining purified bacterial polysaccharides. The purified polysaccharides may be used as antigens, or may be used for production of antibodies, and vaccines. The purified polysaccharides may be used alone or may be conjugated to carrier molecules.

[0065] In an aspect of the present disclosure, there is provided a method for obtaining purified bacterial polysaccharides. The method comprises a single step, wherein impurities such as proteins and nucleic acid are removed from the crude polysaccharides and at the same time the molecular weight of the crude polysaccharide is also reduced to a desired range.

[0066] The method for obtaining the purified bacterial polysaccharides in accordance with the present disclosure comprises incubating/treating a fermentation harvest comprising polysaccharide with an acid at a pre-determined temperature and pH for a pre-determined time period.

[0067] In an embodiment of the present disclosure there is provided a method for obtaining purified and sized bacterial polysaccharides, the method comprising the following steps: [0068] (a) providing a fermentation harvest comprising bacterial cell, polysaccharide, proteins, nucleic acid and cell debris; and [0069] (b) treating the fermentation harvest with an acid to separate the polysaccharide from protein, nucleic acid and cell debris to obtain purified and sized bacterial polysaccharides.

[0070] The pH of the fermentation harvest may be in the range of 5.8 to 6.8.

[0071] The cell debris may contain cell wall polysaccharide (CWPs), which is considered a contaminant in the final product.

[0072] Typically, the bacterial cell in the fermentation harvest is first lysed, using known techniques, to release the polysaccharide. Cell lysis may be carried out using detergents (such as deoxycholate sodium, DOC), enzymes (such as lysozymes), mechanical/physical means (such as sonication), and the like. The fermentation harvest containing the lysed bacterial cells may be then centrifuged and filtered. Centrifugation helps in separation and removal of undesired cell debris, colloids and large aggregates thereby reducing burden on downstream processing. Filtration, such as microfiltration (e.g. 0.2) aids in the removal of fermentation media components. In an embodiment of the present disclosure, post fermentation, cell-free supernatant is subjected to tangential flow filtration (TFF) using a membrane having 100 kDa to 150 kDa molecular weight cut off (MWCO).

[0073] The method of the present disclosure may be used for obtaining purified polysaccharides from any bacteria. In accordance with the embodiments of the present disclosure, the polysaccharides may be derived from bacteria selected from the group consisting of, but not limited to, Streptococcus spp. such as Group A Streptococcus, Group B Streptococcus (group Ia, Ib, II, III, IV, V, VI, VII, VII, VIII, and IX); Streptococcus pneumoniae, Streptococcus pyogenes; Streptococcus agalactiae; Streptococcus viridans; Salmonella spp. such as, Salmonella typhi; Salmonella paratyphi; Salmonella enteritidis; Salmonella typhimurium; Shigella spp.; E. coli; Neisseria meningitidis (serotypes such as A, B, C, E29, H, I, K, L, M, W135, X, Y, Z, etc); Neisseria gonorrhoeae; Haemophilus influenzae; Haemophilus pneumonia; Helicobacter pylori; Chlamydia pneumoniae; Chlamydia trachomatis; Ureaplasma urealyticum; Mycoplasma pneumoniae; Staphylococcus spp. such as Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8; Enterococcus faecalis; Enterococcus faecium; Bacillus anthracis; Vibrio cholerae; Pasteurella pestis; Pseudomonas aeruginosa; Campylobacter spp. such as Campylobacter jejuni; Clostridium spp. such as Clostridium difficile; Mycobacterium spp. such as Mycobacterium tuberculosis; Moraxella catarrhalis; Klebsiella pneumoniae; Treponema spp.; Borrelia spp.; Borrelia burgdorferi; Leptospira spp.; Hemophilus ducreyi; Corynebacterium diphtheria; Bordetella pertussis; Bordetella parapertussis; Bordetella bronchiseptica; Ehrlichia spp.; and Rickettsia spp.

[0074] In an embodiment, the bacterial polysaccharide is derived from Streptococcus pneumoniae serotype selected from the group consisting of, but not limited to, 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48.

[0075] The bacteria used in the present disclosure can be sourced from known collection centres, such as Centers for Disease Control and Prevention (CDC), Atlanta USA; CBER/FDA, USA; ATCC, USA; NIH, USA; NIAID, USA; and PHE, UK. In one embodiment, the Streptococcus pneumoniae serotypes are sourced from CDC, Atlanta, USA.

[0076] Acids that can be used in the present disclosure include, but are not limited to, trifluoroacetic acid, trichloroacetic acid, sulphuric acid, phosphoric acid, acetic acid, hydrochloric acid, nitric acid. In accordance with the embodiments of the present disclosure, the concentration of the acid in the fermentation harvest is in the range of 0.1 M to 20 M, 1 M to 10 M, 1M to 4M, preferably 2 M to 5 M (the concentration mentioned here refers to the final concentration of the acid in the fermentation harvest).

[0077] In an embodiment, the acid is Trifluoroacetic acid (TFA). TFA is a mineral acid with the chemical formula CF.sub.3CO.sub.2H. It is a structural analogue of acetic acid with all three of the acetyl group's hydrogen atoms replaced by fluorine atoms and is a colorless liquid with a vinegar like odor. TFA is a stronger acid than acetic acid, having an acid ionisation constant that is approximately 34,000 times higher, as the highly electronegative fluorine atoms and consequent electron-withdrawing nature of the trifluoromethyl group weakens the oxygen-hydrogen bond (allowing for greater acidity) and stabilises the anionic conjugate base.

[0078] The temperature during acid treatment may be in the range of 2 C. to 100 C., 10 C. to 90 C., 20 C. to 80 C., preferably between 20 C. to 60 C.

[0079] The pH during acid treatment may be less than 2, less than 1.5, less than 1.0, preferably less than 0.5.

[0080] The incubation time period may range from 1 hour to 10 hours, preferably 2 hours to 6 hours.

[0081] The incubation/treatment of the crude polysaccharide with the acid results in the precipitation of the impurities, namely, proteins and nucleic acids. The precipitated impurities are optionally removed by centrifugation. The centrifugation may be carried out between 8000 g to 18000 g, at a temperature between 4 C. to 10 C. for a time period of 10 minutes to 30 minutes. In an embodiment, the supernatant may be further incubated as per the parameters described hereinabove to remove any residual impurities present and to obtain the desired molecular size of the polysaccharide.

[0082] The method of the present disclosure is capable of simultaneous purification and sizing of polysaccharides. It is observed that the impurities including protein, nucleic acid and CWPs are removed during the treatment with the acid. Sizing/depolymerization of the polysaccharide is also initiated at this time. The resulting mixture containing cell debris and impurities is centrifuged to separate the impurities. The supernatant is then further incubated to obtain polysaccharide having the desired molecular size. However, there is no further addition of TFA at this stage; rather the TFA present in the supernatant is capable of sizing/depolymerizing the polysaccharides to the desired molecular size. The residual TFA is removed from the final polysaccharide during the subsequent diafiltration step. In order to cease the action of TFA, dibasic sodium is added prior to the diafiltration step.

[0083] The bacterial polysaccharide is periodically monitored to ascertain/determine the molecular size. Typically, chromatographic techniques, such as Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) are used to ascertain/determine the molecular size of the bacterial polysaccharide. Typically, the average molecular size of the bacterial polysaccharide after the incubation/treatment with TFA is in the range of 10 kDa to 1000 kDa, 50 kDa to 600 kDa, 50 kDa to 300 kDa, 50 kDa to 250 kDa, preferably 100 to 200 kDa. In an exemplary embodiment of the present disclosure, the average molecular size of the purified bacterial polysaccharide is in the range of 50 kDa to 200 kDa. Residual acid may be removed during the subsequent diafiltration steps.

[0084] Conventionally, the polysaccharides are sized to the desirable molecular weight in a separate step using techniques, such as microwave, sonication, microfluidization, and high pressure homogenization. However, microwave technique results in high consumption of power and also the polysaccharide structure is easy to be destroyed. High pressure homogenization is expensive; results in generation of heat, which could damage the product of interest; and also further time-consuming methods are required to purify the product. Sonication results in generation of heat, which may degrade the product of interest. On the other hand, the method of the present disclosure employs simultaneous purification and sizing of the polysaccharides, and thereby saving time, labour and cost.

[0085] After the incubation/treatment is over, the pH of the mixture is raised to be in the range of 5.5 to 6.5 using a pH adjusting agent. In an embodiment of the present disclosure, the pH adjusting agent is disodium hydrogen phosphate (Na.sub.2HPO.sub.4). The pH adjusting agent is slowly added to the mixture with agitation to raise the pH. Other pH adjusting agents, such as dipotassium hydrogen phosphate (K2HPO.sub.4) are also envisaged by the method of the present disclosure.

[0086] Once the pH of the mixture is maintained in the range of 5.5 to 6.5, the mixture is subjected to diafiltration in the presence of a suitable diluent using a membrane having 5 kDa to 30 kDa a molecular weight cut off (MWCO) to obtain the purified and sized bacterial polysaccharide. In a preferred embodiment, diafiltration is carried out using a 10 kDa a MWCO. In another preferred embodiment, the diluent is water for injection. In an embodiment, the purified bacterial polysaccharide has a pH in the range of 6.0 to 7.0.

[0087] Typically, the recovery of purified polysaccharides using the method of the present disclosure is more than 60%.

[0088] Further, the method of the present disclosure is capable of substantially reducing impurities from the bacterial polysaccharide. In an embodiment, the impurities are reduced to, i.e., proteins to less than 3% and nucleic acids to less than 2%. In a preferred embodiment, the protein content is reduced to less than 2% and the nucleic acid is reduced to less than 0.5%. Still further, the purified and sized bacterial polysaccharide obtained by the process of the present disclosure has polysaccharide polydispersity less than 2 and CWPs content not more than 2 mol %.

[0089] In the present disclosure, it is surprisingly found that TFA is capable of removing the impurities from the crude polysaccharides and also size the polysaccharide to a range of 10 kDa to 1000 kDa by controlling parameters, such as pH and concentration of TFA, reaction time and temperature of the reaction.

[0090] Conventional processes for obtaining purified bacterial polysaccharide are time consuming, multi-step and labour intensive, using enzyme treatments, single/multiple chromatographic steps, tangential flow filtration (TFF) systems/cassettes. This is followed by separate sizing process involving physical, chemical or mechanical means. Whereas, the method of the present disclosure is a single step process using TFA resulting in simultaneous removal of impurities as well as sizing of the crude polysaccharide in the desirable range. Also, the use of TFA aids in the lysis of the bacterial cell, thereby releasing the polysaccharide, which is then further purified and sized to obtain the purified polysaccharide. The method of the present disclosure also avoids chemicals, such as CTAB, alcohol, and ammonium sulphate; enzymatic treatment, and chromatographic steps, whereby further labour intensive and expensive down stream processing steps are prevented. Hence, the method of the present disclosure is simple, cost effective, rapid, and provides high recovery of the polysaccharide.

[0091] In an embodiment of the present disclosure, the method or producing purified and sized bacterial polysaccharides does not use a lytic agent and comprises the following steps: [0092] (a) providing a fermentation harvest comprising bacterial cell; [0093] (b) subjecting the fermentation harvest to centrifugation to separate cell free supernatant; [0094] (c) concentrating and filtering the cell free supernatant of step (b); [0095] (d) treating the concentrated and filtered cell free supernatant of step (c) with an acid having concentration in the range of 2 M to 5M at 20 C. to 60 C. and pH in the range of 0.1 to 2; [0096] (e) subjecting the acid treated mixture of step (d) to centrifugation to remove precipitated impurities; [0097] (f) incubating the supernatant of step (e) at a temperature in the range of 20 C. to 50 C.; [0098] (g) raising the pH of the solution of step (f) using a pH adjusting agent; [0099] (h) diafiltering the solution of step (g) to increase the concentration of polysaccharide in the solution; and [0100] (i) filtering the concentrated polysaccharide of step (h) to obtain purified and sized polysaccharide having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, polydispersity less than 2, molecular size in the range of 10 kDa to 600 kDa (SEC-HPLC), and >10% methyl pentose content.

[0101] After fermentation, the fermentation harvest is centrifuged to remove the cell debris, this is called as cell free supernatant. In order to remove the media components the cell free supernatant is further diafiltered using 100 kDa cutoff membrane. This media and cell free supernatant called as 100 kDa crude polysaccharide and is used for further processing.

[0102] Typically, the filtration in step (c) is carried out on 100 kDa tangential flow filtration (TFF) membrane at a pH in the range of 5.5 to 8 and the filtration in step (h) is carried out on 10 kDa tangential flow filtration (TFF) membrane at a pH in the range of 4 to 6. The filtration in step (i) is carried out using 0.2 filter at a pH in the range of 6 to 7.

[0103] Cells may be in the form of a wet cell paste or are suspended in an aqueous medium.

[0104] In another embodiment of the present disclosure, the method or producing purified and sized bacterial polysaccharides uses a lytic agent. Typically, the fermentation harvest is treated with a lytic agent prior to treatment with an acid.

[0105] In accordance with the embodiments of the present disclosure, the lytic agent can be selected from mechanical and non-mechanical lytic agents.

[0106] The mechanical lytic agent can be selected from sonication, high pressure homogenization and bead mill.

[0107] In one embodiment, the non-mechanical lytic agent is a chemical lytic agent selected from alkali and detergents. Typically, the chemical lytic agent can be selected from the group consisting of deoxycholate sodium (DOC), sodium dodecyl sulphate (SDS), decanesulfonic acid, tert-octylphenoxy poly(oxy ethylene)ethanols, octylphenol ethylene oxide condensates, N-lauryl sarcosine sodium (NLS), lauryl iminodipropionate, chenodeoxycholate, hyodeoxycholate, glycodeoxycholate, taurodeoxycholate, taurochenodeoxycholate, cholate, triton X, Tween (20, 80), EDTA, urea and cetyltrimethylammonium bromide (CTAB).

[0108] In another embodiment, the non-mechanical lytic agent can be a physical lytic agent selected from thermal lysis, cavitation and osmotic shock.

[0109] In still another embodiment, the non-mechanical lytic agent can be an enzyme selected from lysozyme, lysostaphin, zymolase, proteinase K, protease and glycanase. In an embodiment of the present disclosure, the method for producing purified and sized bacterial polysaccharides comprises the following steps: [0110] (a) providing a fermentation harvest comprising bacterial cell; [0111] (b) treating the fermentation harvest with a lytic agent to obtain a mixture of polysaccharide, proteins, nucleic acid and cell debris; [0112] (c) subjecting the lytic agent treated fermentation harvest of step (b) to centrifugation to separate cell free supernatant; [0113] (d) concentrating and filtering the cell free supernatant of step (c); [0114] (e) treating the concentrated and filtered cell free supernatant of step (d) with an acid having concentration in the range of 2 M to 5M at 20 C. to 60 C. and pH in the range of 0.1 to 2; [0115] (f) subjecting the acid treated mixture of step (e) to centrifugation to remove precipitated impurities; [0116] (g) incubating the supernatant of step (f) at a temperature in the range of 20 C. to 50 C.; [0117] (h) raising the pH of the solution of step (g) using a pH adjusting agent; [0118] (i) diafiltering the solution of step (h) to increase the concentration of polysaccharide in the solution; and [0119] (j) filtering the concentrated polysaccharide of step (i) to obtain purified and sized polysaccharide having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, polydispersity less than 2, molecular size in the range of 10 kDa to 600 kDa (SEC-HPLC), and >10% methyl pentose content.

[0120] Typically, the step of cell lysis is carried out at a pH in the range of 5 to 7.

[0121] After fermentation, the treatment with the lytic agent is carried out for to lysis of cells. The fermentation harvest is then centrifuged to remove the cell debris, this is called as cell free supernatant. In order to remove the media components the cell free supernatant is further diafiltered using 100 kDa cutoff membrane. This media and cell free supernatant called as 100 kDa crude polysaccharide and is used for further processing.

[0122] The filtration in step (d) is carried out on 100 kDa tangential flow filtration (TFF) membrane at a pH in the range of 5.5 to 8 and the filtration in step (i) is carried out on 10 kDa tangential flow filtration (TFF) membrane at a pH in the range of 4 to 6. The filtration in step (j) is carried out using 0.2 filter.

[0123] The present disclosure further envisages the use of at least one chromatography step to obtain the purified and sized polysaccharide. The chromatography can be selected from the group consisting of ion-exchange (cationic or anionic), affinity chromatography, hydrophilic-interaction, hydrophobic-interaction, size-exclusion, hydroxyl apatite, gel-permeation chromatography, Cibacron Blue pseudo affinity sorbent, mixed mode chromatography sorbent, membrane chromatography, Capto Adhere, TOYOPEARL MX-Trp-650M, Cellufine MAX AminoButyl, monolith chromatography device, adsorbent chromatography, lectin agarose column, and Amberlite column.

[0124] Further, the method of the present disclosure may comprise a step of treatment with one or more chemical/biological reagent at any stage of the method. The reagent can be selected from the group consisting of cetyl trimethyl ammonium bromide (CTAB), hexadimethrine bromide and myristyltrimethylammonium, triton, acetate, sodium carbonate, zinc, enzymes, alcohol (ethanol, isopropanol), phenol, acetone, salts (magnesium, calcium), sodium dodecyl sulfate (SDS), polysorbates, sodium sarcosine, NaCl, urea, formaldehyde, ammonium chloride, ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, mineral acid, organic acid, metal cations, toluene, chloroform, alkali, ascorbic acid, tetrabutylammonium, potassium chloride, alkyl sulfates, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium -olefin sulfonates, sodium alkyl sulfonates, -sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, sodium alkyl glyceryl ether sulfonates, DNase and/or RNase, Benzonase, Mutanolysin/lysozyme, beta-D-N-acetyl glucosaminidase, and Proteinase K.

[0125] The purified and sized polysaccharide may be subjected to an additional size reduction step selected from thermal treatment, sonic treatment, chemical hydrolysis, endolytic enzyme treatment, and physical shear, Gaulin-homogenizer, and sonication.

[0126] In an embodiment, there is provided a bacterial polysaccharide obtained by the method of the present disclosure. The polysaccharide thus obtained has protein content less than 3%, nucleic acid content less than 2%, molecular size in the range of 10 kDa to 600 kDa (SEC-HPLC), and polysaccharide recovery of at least 60%.

[0127] The purified and sized polysaccharide obtained by the method of the present disclosure may be used as antigens, or may be used for production of antibodies, and vaccines (plain polysaccharide or polysaccharide-protein conjugate). The purified polysaccharides may be used alone or may be conjugated to carrier molecules.

[0128] In an embodiment, there is provided an immunogenic composition comprising Streptococcus pneumoniae polysaccharides, wherein the polysaccharide is obtained by the method of the present disclosure having protein content less 3%, nucleic acid content less than 2%, molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC) and wherein the polysaccharide is derived from Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32A, 32F, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, and 48.

[0129] In accordance with the embodiments of the present disclosure, the immunogenic composition is a 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30-valent pneumococcal polysaccharide-protein conjugate composition.

[0130] In an embodiment, the immunogenic composition comprises polysaccharides that are conjugated to carrier protein selected from CRM197, tetanus toxoid (TT), diphtheria toxoid (DT), Neisseria meningitidis outer membrane complex, fragment C of tetanus toxoid, pertussis toxoid, protein D of H. influenzae, E. coli LT, E. coli ST, and exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified protein derivative of tuberculin (PPD), cholera toxin B, synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins or any equivalents thereof.

[0131] In an embodiment, there is provided an immunogenic composition comprising 10 distinct Streptococcus pneumoniae polysaccharide-protein conjugates wherein [0132] the polysaccharide is obtained by the method of the present disclosure having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC); [0133] the polysaccharides are derived from serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F; and [0134] all the polysaccharides are conjugated to CRM197 as the carrier protein.

[0135] In another embodiment, there is provided an immunogenic composition comprising 17 distinct Streptococcus pneumoniae polysaccharide-protein conjugates wherein [0136] the polysaccharide is obtained by the method of the present disclosure having protein content less than 3%, nucleic acid content less than 2%, CWPs content not more than 2 mol %, molecular size in the range of 10 kDa to 300 kDa (SEC-HPLC); [0137] the polysaccharides are derived from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F and 23F; and [0138] serotype 3 is conjugated to CRM197, serotype 18C is conjugated to CRM197, serotype 4 is conjugated to DT, serotype 15B is conjugated to TT and serotype 22F is conjugated to TT.

[0139] The polysaccharides can be conjugated to the carrier protein using known conjugation chemistry, such as cyanylation chemistry, CNBr chemistry, reductive amination chemistry, and carbodiimide chemistry.

[0140] Non-limiting examples of cyanylation agent include 1-cyano-4-dimethylaminopyridinium tetrafluroborate (CDAP), 1-cyano-4-pyrrolidinopyridinium tetrafluorborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 2-cyanopyridazine-3(2H)one (2-CPO), and a functional derivative or modification thereof.

[0141] In one embodiment, the polysaccharide can be conjugated to the carrier protein in the presence of a linker selected from the group comprising hexanediamine, ethylene diamine, hydrazine, adipic dihydrazide, and 1,6-diaminooxyhexane.

[0142] In another embodiment, the polysaccharide can be conjugated to the carrier protein in the absence of a linker.

[0143] The immunogenic composition may further comprise pharmaceutically acceptable excipients selected from surfactants, stabilizers, buffers, diluents, adjuvants, preservatives and solvents.

[0144] In another embodiment, polysaccharide-protein conjugate vaccine compositions prepared using the polysaccharides of the present disclosure are multi-dose compositions comprising at least one preservative. The preservative may be selected from the group comprising 2-phenoxyethanol, benzethonium chloride (Phemerol), phenol, m-cresol, thiomersal, formaldehyde, methyl paraben, propyl paraben, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, benzyl alcohol and combinations thereof.

[0145] One of the critical parameters for immunogenicity for a number of Streptococcus pneumoniae serotypes is the amount of methyl pentose groups in the polysaccharide. Conventional processes fail to retain the required amount of methyl pentose in the polysaccharides. However, it is observed that the method of present disclosure is capable of preserving the methyl pentose groups present in the polysaccharide of certain serotypes of Streptococcus pneumoniae as required by the WHO guidelines.

[0146] Typically, the methyl pentose content for Streptococcus pneumoniae serotypes 2, 4, 5, 6B, 7F, 12F, 17A, 17F, 19A, 19F and 23F obtained by the method of the present disclosure is 10%. Specifically, serotype 2 has a methyl pentose content of at least 50%, serotype 18C has a methyl pentose content of at least 25% and serotype 22F has a methyl pentose content of at least 35%.

[0147] In another embodiment, the method of the present disclosure is capable of preserving the desired amount of other groups critical for immunogenicity, such as pyruvates, uronic acid, hexosamine, glycerol, acetate and O-acetyl groups. In one embodiment, the acetate content in the polysaccharides for serotypes 22F and 15B is about 1 mM per mM of the respective polysaccharide. In another embodiment, the glycerol content in the polysaccharide 15B is about 1 mM per mM of the polysaccharide.

[0148] Further, it is seen that the free polysaccharide and free protein content of polysaccharide-carrier protein conjugates prepared using polysaccharides obtained by the method of present disclosure are within the specified limit and hence conjugates are found to be stable.

[0149] The present disclosure is further described in light of the following examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.

EXAMPLES

[0150] The Streptococcus pneumoniae serotypes 2, 18C and 22F were sourced from Centers for Disease Control and Prevention (CDC), Atlanta, USA.

[0151] U.S. Pat. No. 9,249,439 is being incorporated with reference to the fermentation processes utilized for all S. pneumoniae serotypes

Example-1a: Obtaining Purified and Sized Polysaccharide from Streptococcus pneumoniae Serotype 2 [Using DOC]

[0152] Sized and purified polysaccharide from Streptococcus pneumoniae serotype 2 was obtained as per the method illustrated in Table-1a.

TABLE-US-00001 TABLE-1a Parameters S. Reagent/ Temperature Time No. Step Instrument used pH ( C.) (Minutes) 1. Fermentation SS Fermenter 20L 7.1 36.5 360 [Agitation 150 RPM & working 0.2 vvm aeration] 2. Cell Lysis (DOC) In SS Fermenter 6.3 15 840 (From 12% stock 310 mL added to get final 2.5% DOC conc.) 3. Cell free supernatant Batch mode 15 45 centrifugation (12000 RCF, 45 minutes, 15 C.) 4. Diafiltration (DF) & 100 kDa TFF 6.5 RT 180 concentration 5. TFA treatment Conc. TFA (final 0.2 40 60 [PS sizing initiate & conc. 3.24M) impurities precipitated] (e.g. 100 mL reaction mixture 25 mL conc. TFA + 75 mL crude PS) 6. Centrifugation Batch mode cent. 12 30 [To remove the (15000 RCF, 30 precipitate] minutes, 12 deg. C.) 7. Incubation In water bath 40 480 [OR incubation continued till the desired molecular weight was reached] 8. Termination of Di sodium 5.5 RT 20 reaction hydrogen [pH adjustment before phosphate (solid DF & concentration] added as per Q.R.) (For 100 mL reaction mixture 55 g added, which is equal to 3.8M) 9. Diafiltration & 10 kDa TFF RT 300 concentration [DF continued till the condition of permeates is equal to WFI conductivity] 10. Final filtration 0.2 micron filter 6.5 RT 15 (Depends on the batch size) Abbreviations: RTroom temperature, DOCDeoxycholic acid, DFdiafiltration, TFFtangential flow filtration, kDakilo Dalton, PSpolysaccharides, TFAtrifluoroacetic acid, RCFRelative Centrifugal Force, Q.R.quantity required, WFIwater for injection

Example-1b: Obtaining Purified and Sized Polysaccharide from Streptococcus pneumoniae Serotype 18C [Using DOC]

[0153] Sized and purified polysaccharide from Streptococcus pneumoniae serotype 18C was obtained as per the method illustrated in Table-1b.

TABLE-US-00002 TABLE-1b parameters S. Reagent/ Temperature Time No. Step Instrument used pH ( C.) (Minutes) 1. Fermentation SS Fermenter 20L 7.1 36.5 345 [Agitation 100 RPM & working 0.2 vvm aeration] 2. Cell Lysis (DOC) In SS Fermenter 6.3 15 840 (From 12% stock 530 mL added to get final 2.5% DOC conc.) 3. Cell free supernatant Batch mode cent. 15 45 (12000 RCF, 45 minutes, 15 C.) 4. Diafiltration (DF) & 100 kDa TFF 6.1 RT 180 concentration 5. TFA treatment 50% TFA as per 0.5 30 60 [PS sizing initiate & Q.R. (e.g. 100 mL impurities precipitated] PS + 45 mL 50% diluted TFA added (final conc. 2.92M)) 6. Centrifugation Batch mode cent. 12 30 [To remove the (15000 RCF, 30 precipitate] minutes, 12 C.) 7. Incubation In water bath 30 1440 OR incubation continued till the desired molecular weight was reached] 8. Termination of reaction Di sodium 5.5 RT 20 [pH adjustment before hydrogen DF & concentration] phosphate (For 100 mL reaction mixture 25 g added, which is equal to 1.73 M ) 9. DF & concentration 10 kDa TFF RT 300 [DF continued till the condition of permeates is equal to WFI conductivity] 10. Final filtration 0.2 micron filter 6.2 RT 15 (Depends on the batch size) Abbreviations: RT room temperature, DOCDeoxycholic acid, DFdiafiltration, TFFtangential flow filtration, kDakilo Dalton, PSpolysaccharides, TFAtrifluoroacetic acid, RCFRelative Centrifugal Force, Q.R.quantity required, WFIwater for injection

Example-1c: Obtaining Purified and Sized Polysaccharide from Streptococcus pneumoniae Serotype 22F [Using DOC]

[0154] Sized and purified polysaccharide from Streptococcus pneumoniae serotype 22F was obtained as per the method illustrated in Table-1c.

TABLE-US-00003 TABLE-1c Parameters S. Reagent/Instrument Temperature Time No. Step used pH ( C.) (Minutes) 1. Fermentation SS Fermenter 20L 7.1 36.5 360 [Agitation 100 RPM & working 0.2 vvm aeration] 2. Cell Lysis (DOC) In SS Fermenter (From 6.3 15 840 12% stock 520 mL added to get final 2.5% DOC conc.) 3. Cell free supernatant Batch mode cent. 15 45 (15000 RCF, 45 minutes, 15 C.) 4. Diafiltration (DF) & 100 kDa TFF 6.7 RT 180 concentration 5. TFA treatment Conc. TFA (final conc. 0.2 30 60 [PS sizing initiate & 3.24M) (In 100 mL impurities reaction mixture 25 mL precipitated] conc. TFA + 75 mL crude PS) 6. Centrifugation Batch mode cent. 12 30 [To remove the (12000 RCF, 30 min., precipitate] 12 deg. C.) 7. Incubation In water bath 30 300 [OR incubation continued till the desired molecular weight was reached] 8. Termination of Di sodium hydrogen 5.8 RT 20 reaction phosphate (solid added [pH adjustment before as per Q.R.) (For 100 DF & concentration] mL reaction mixture 55 g added, which is equal to 3.8M) 9. Diafiltration & 10 kDa TFF RT 300 concentration [DF continued till the condition of permeates is equal to WFI conductivity] 10. Final filtration 0.2 micron filter 6.6 RT 15 (Depends on the batch size) Abbreviations: RTroom temperature, DOCDeoxycholic acid, DFdiafiltration, TFFtangential flow filtration, kDakilo Dalton, PSpolysaccharides, TFAtrifluoroacetic acid, RCFRelative Centrifugal Force, Q.R.quantity required, WFIwater for injection

[0155] Tables (1a, 1b and 1c) provide the process steps and parameters for fermentation of S. pneumoniae serotypes 2, 18C and 22F along with the steps for purification and sizing of the polysaccharides using trifluoroacetic acid (TFA).

Example-2: Effect of TFA on Polysaccharide

[0156] The polysaccharides obtained in Examples 1a, 1b and 1c were analysed and the result obtained is illustrated in Table-2.

TABLE-US-00004 TABLE 2 Impurities profile Mw by % SEC-HPLC % Nucleic Immuno- % CWPs Character- Serotype (kDa) Protein acid reactivity Yield (from NMR) Proton NMR ization 2 153 0.25 0.3 121% 87 2 mol % Identity & Methyl integrity pentose 57% matched with (limit 38%) 18C 185 1.4 0.3 322% 78 <1.5 mol % standard Methyl pentose 28% (limit 14%) 22F 154 0.32 0.16 157% 71 <1.9 mol % Methyl pentose 38% (limit 25%)

[0157] It is seen from Table-2 that the molecular size (SEC-HPLC) for polysaccharides of S. pneumoniae serotypes 2, 18C and 22F obtained by the method of present method is below 200 kDa, the protein content is less than 2%, the nucleic acid content is less than 0.3%, polysaccharide yield is more than 70%, immunoreactivity is more than 100%, and CWPs is not more than 2 mol %. Also the methyl pentose content is within the specified limit.

[0158] Different concentrations of antigen were neutralized with antigen specific antibodies and mixture was added to antigen coupled beads. The remaining unneutralized antibody binds to the coupled onto the beads. Antibody bound to beads was detected by anti mouse phycoerythrin conjugate. Quantitation of antigen was inversely proportional to median fluorescence intensity (MFI) generated by the reaction. Higher the antigen contains, lower the MFI and vice versa. WO2013046226A2 is being incorporated with reference to the method used for determining immunoreactivity for the polysaccharides.

[0159] NMR analysis of the purified polysaccharides was carried out and the results obtained are illustrated in FIGS. 1-7.

[0160] 1D spectrum recorded at 323 K contains a sharp signals from O-acetylated S. pneumoniae polysaccharide from serotype 22F RU together with small peaks from CWPs. The O-acetylated 22F RU was identified by key signals in the anomeric (six H1 and H2 of Rha2Ac), acetyl (O-acetyl) and methyl (H6 of - and -Rha) regions. This was confirmed by 1H NMR analysis using the peak area of H6 of - and -Rha, the mole percent of CWPs were determined as 1.2 mol %. Integration of O-acetyl signal shows the sample has 86% O-acetylation, from the rest of the spectrum it appears to be 100%. The structure of the specific capsular polysaccharide produced by Streptococcus pneumoniae type 22F was investigated by 1H nuclear magnetic resonance spectroscopy. The polysaccharide was found to be a high molecular weight acidic polymer composed of D-glucose, D-galactose, D-glucuronic acid, and L-rhamnose residues to form a regular repeating hexasaccharide unit having the above structure in which the -L-rhamnopyranosyl residues were substituted by O-acetyl groups in 80% of the repeating units (FIGS. 1-2).

[0161] Pn 18C identity was confirmed by 1 D NMR experiments and literature. P-31 NMR gave major

phosphodiester and three CWPs signals which means 2 Cho per CWPs, i.e., 18H for calculation. Low level CWPs was found. By NMR integration of H6 of Rha compared to H2 of Glc, estimate 1.4 mole of methyl pentose per RU. CWPs mole percent is 1.45% from integration of .sup.1H NMR (FIGS. 3-5)

[0162] From the literature Pn 2 polysaccharide (FIGS. 6a-6b) consists of a hexasaccharide repeating unit and it gives broad lines due to the rigid RU structure (a doubly-branched sugar) and high viscosity. Some proton literature assignments have been published in Carbohydrate Research, 1988.

[0163] NMR spectra recorded at 323 K (Figure-7) showed diagnostic signals from the six H1 signals, H6 signals from Rha (3) and the presence of low amounts of CWPs. The P-31 spectrum gave 3 signals consistent with the presence of two PCho grows. Integration of the CWPs signal and H6 of Rha gave a CWPs of 2% mole percent. Thus the structure of the RU is consistent with literature, however, full proton and carbon assignments (not available in the literature) will require additional 2D NMR experiments to be performed.

[0164] Similar results were obtained when purified and sized polysaccharides were obtained in the absence of lytic agent (DOC).

[0165] The method of the present disclosure is capable of simultaneous purification and sizing of polysaccharides. It was observed that the impurities including protein, nucleic acid, CWPs are removed during the treatment with the acid. Sizing/depolymerization of the polysaccharide is also initiated at this time. The resulting mixture containing cell debris and impurities was centrifuged to separate the impurities. The supernatant was then further incubated to obtain polysaccharide having the desired molecular size. However, there was no further addition of TFA at this stage; rather the TFA present in the supernatant was capable of sizing/depolymerizing the polysaccharides to the desired molecular size. The residual TFA was removed from the final polysaccharide during the subsequent diafiltration step.

[0166] Residual TFA was analysed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) method. It was observed that in the final purified polysaccharide the concentration of residual TFA in per mL sample is 2 ppb, which illustrates that the TFA was removed from product (polysaccharide) during the diafiltration step.

Following Precautions/Process are to be Taken while Handling TFA: [0167] 1) Person shall wear all safety kit, such as acid proof hand gloves, goggles, face shield, etc. while performing operation(s) with TFA. [0168] 2) Operation(s) with TFA must have performed under the controlled environment such as air flow unit(s), such as hanging fan flow unit/fume hood, etc. [0169] 3) Acid resistant/proof equipment(s) shall be considered for scale up the process to the industrial level as the pH of the reaction mixture will be below 1.0. Thereafter all the subsequent operation(s) are carried out at normal pH, hence no special equipment(s) are required.

Example-3: Comparison of Effect of the Method of Present Disclosure and Conventional Process [Homogenization+HIC+IEC] for Serotypes 2, 18C and 22F

[0170]

TABLE-US-00005 TABLE 3 TFA with DOC HIC + IEC + Homogenization Experimental data Serotype Serotype Serotype Serotype Serotype Serotype S. No. specifications 2 18C 22F 2 18C 22F 1) Polysaccharide Mw 153 185 154 157 163 145 by SEC-HPLC (kDa) 2) Polysaccharide 1.4 1.5 1.4 1.8 1.92 1.9 polydispersity 3) Polysaccharide % 87 78 71 66 70 68 yield/recovery 4) % protein impurity 0.1 0.2 0.1 0.25 1.4 0.32 5) % nucleic acid ND 0.1 ND 0.3 0.1 0.16 impurity 6) CWPs impurity 2 mol % <1.5 mol % <1.9 mol % >6 mol % >6 mol % >6 mol % content (by NMR) 7) Conjugate stability Stable Stable Stable Stable Stable Stable 8) Conjugate Passes Passes Passes Passes Passes Passes immunogenicity 9) Free Ps (%) 2.55 15.32 <1 4.84 17.11 3.81 10) Free protein (%) <2 <2 ND <4 <5 <1 ND = Not detected

[0171] It is seen from Table-3 that the molecular size (SEC-HPLC) for polysaccharides of S. pneumoniae serotypes 2, 18C and 22F obtained by the method of present method is below 200 kDa, the protein content is less than 1%, the nucleic acid content is less than 0.3%, polysaccharide yield is more than 70%, and CWPs is not more than 2%. It is seen from Table-3 that the method of present disclosure is capable of producing polysaccharides with higher yield when compared with conventional methods. Also, the method of present disclosure is results in comparatively lower CPS impurity.

[0172] Further, it is seen from Table-3 that the free protein and free polysaccharide content for the conjugates prepared using the polysaccharides prepared in accordance with the method of present disclosure is within the specified limits and hence the conjugates are stable.

[0173] Similar results were obtained when purified and sized polysaccharides were obtained in the absence of lytic agent (DOC).

[0174] U.S. Ser. No. 10/729,780 is being incorporated with reference to the polysaccharide protein conjugation processes and multivalent S. pneumoniae conjugate formulations (10 valent, 17 valent) obtained thereof.

[0175] The method of present disclosure for simultaneous purification and sizing/depolymerization of polysaccharide by TFA provides numerous advantages over the conventional process. The method of present disclosure is devoid of processes, such as benzonase treatment, ammonium sulphate precipitation, HIC, IEC and homogenization, thereby making the present method rapid, less labor intensive, cost effective, and reduces the loss of yield during the downstream processing. The polysaccharides obtained by the method of the present disclosure, and the conjugates prepared therefrom meets the specifications as per monographs IP/BP/USP/WHO and are stable over extended periods of time.

Technical Advantages

[0176] The method for obtaining purified and sized bacterial polysaccharides of the present disclosure described herein above has several technical advantages including, but not limited to, the realization of: [0177] A single step method for purification of bacterial polysaccharides with high recovery, low impurity content [0178] Optimal preservation of integrity of polysaccharide (in terms of methyl pentose, acetate and glycerol content) thereby ensuring stable and immunogenic conjugates; [0179] Avoiding a separate step for mechanical sizing of polysaccharides; [0180] Reduction in process time for purification of crude polysaccharides by about 60%; [0181] Simultaneous removal of impurities as well as sizing of bacterial polysaccharides; and [0182] Simple and cost effective method for purification of bacterial polysaccharides that does not utilize phenol, triton, enzymes, CTAB, activated carbon, chromatography, ammonium sulphate or ethanol.

[0183] The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

[0184] Throughout this specification the word comprise, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0185] The use of the expression at least or at least one suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired object or results.

[0186] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[0187] The numerical values mentioned for the various physical parameters, dimensions or quantities are only approximations and it is envisaged that the values ten percent higher/lower than the numerical values assigned to the parameters, dimensions or quantities fall within the scope of the disclosure, unless there is a statement in the specification specific to the contrary.

[0188] While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.