METHODS FOR SIMULTANEOUS FRAGMENTATION AND PURIFICATION OF BACTERIAL POLYSACCHARIDES

Abstract

The present disclosure relates to alternative, cost effective, rapid and simple methods for bacterial capsular polysaccharide (CPS) manufacturing resulting in 1) simultaneous sizing and purification of CPS 2) high CPS yield, 3) improved CPS purity and removal of protein and nucleic acid contaminants, 4) CPS with preserved epitopic conformation and 5) stable and immunogenic polysaccharide-protein conjugate vaccines comprising of said size reduced and purified CPS The method particularly comprises subjecting crude/native bacterial polysaccharide to an oxidizing agent to obtain high purity, high yield and structurally intact CPS having optimal molecular size and other desirable CPS attributes. The method is amenable for commercial scale manufacturing of polysaccharide-protein conjugate vaccines.

Claims

1. A method for simultaneous fragmentation and purification of bacterial polysaccharides, the method comprising the following steps: (a) providing a fermentation harvest comprising bacterial cells expressing a polysaccharide; (b) inactivating the bacterial cells in the fermentation harvest by adding an inactivating agent to the fermentation harvest; (c) subjecting the fermentation harvest comprising the inactivated bacterial cells 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 oxidizing agent having a final concentration in the range of 0.5% to 50% at a temperature in the range of 4° C. to 80° C. and incubating for a time period in the range of 2 hours to 15 hours; (f) subjecting the oxidizing agent treated mixture of step (e) to centrifugation to remove precipitated impurities and collect supernatant; and (g) diafiltering and concentrating the supernatant of step (f) to obtain a purified and sized polysaccharide having a molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), a polydispersity in the range of 1 to 5, a hexosamine content between 10% and 55%, a pyruvate content greater than 10%, a protein content less than 2%, a nucleic acid content less than 2%, an immunoreactivity in the range of 40% to 95%, and a percentage yield of polysaccharide in the range of 60% to 95%.

2. The method as claimed in claim 1, wherein the bacterial polysaccharide is 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.

3. The method as claimed in claim 2, wherein the bacterial polysaccharide is 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.

4. The method as claimed in claim 2, wherein the bacterial polysaccharide is derived from Streptococcus pneumoniae serotype selected from the group consisting of 1, 2, 3, 4, 5, 6, 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, 18, 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.

5. The method as claimed in claim 2, wherein the bacterial polysaccharide is derived from a Salmonella spp. selected from the group comprising Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, and Salmonella typhimurium.

6. The method as claimed in claim 2, wherein the bacterial polysaccharide is 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.

7. The method as claimed in claim 2, wherein the bacterial polysaccharide is derived from a Staphylococcus spp. selected from the group comprising Staphylococcus aureus, Staphylococcus aureus type 5, and Staphylococcus aureus type 8.

8. The method as claimed in claim 1, wherein the pH of the fermentation harvest is in the range of 3 to 9, preferably 5 to 8.

9. The method as claimed in claim 1, wherein the oxidizing agent is a peroxide selected from the group comprising peroxy acids, metal peroxides, and organic peroxide.

10. The method as claimed in claim 9, wherein the oxidizing agent is selected from the group consisting of peracetic acid, H.sub.2O.sub.2, barium peroxide, sodium peroxide, and tert-butylhydroperoxide.

11. The method as claimed in claim 1, wherein the concentration of the oxidizing agent is in the range of 2% to 50%, preferably 2% to 20%, more preferably 5 to 10%.

12. (canceled)

13. (canceled)

14. The method as claimed in claim 1, wherein the temperature during step (e) is in the range of 4° C. to 70° C., preferably 10° C. to 40° C., more preferably 15° C. to 35° C., and most preferably 15° C. to 35° C.

15. (canceled)

16. (canceled)

17. The method as claimed in claim 14, wherein the temperature during step (e) is in the range of 25° C. to 35° C., preferably 28° C. to 32° C.

18. (canceled)

19. The method as claimed in claim 1, wherein step (e) is carried out in the presence of a reagent selected from the group consisting of buffers, solvents, metal salts, organic acids and mineral acids.

20. The method as claimed in claim 19, wherein the buffer is selected from the group consisting of TRIS, sodium phosphate, sodium acetate; the organic acid is selected from the group consisting of ascorbic acid, acetic acid, citric acid; and the mineral acid is selected from the group consisting of HCl, H.sub.2SO.sub.4, TFA, and HNO.sub.3.

21. The method as claimed in claim 1, wherein the molecular size of the purified and sized bacterial polysaccharides is in the range of 50 kDa to 600 kDa, preferably 50 kDa to 300 kDa.

22. (canceled)

23. The method as claimed in claim 1, wherein the inactivating agent is selected from the group consisting of detergents, enzymes, mechanical inactivating agents and physical inactivating agents.

24. The method as claimed in claim 1, wherein the inactivating agent is selected from the group consisting of deoxycholate sodium (DOC), lysozymes, and sonication.

25. The method as claimed in claim 1, wherein the centrifugation in step (c) is carried out at 10,000 g to 20,000 g and at a temperature in the range of 10° C. to 20° C.

26. The method as claimed in claim 1, wherein the pH of the supernatant obtained in step (c) is in the range of 5 to 7.

27. The method as claimed in claim 1, wherein the filtration in step (d) is carried out on 100 kDa ultrafiltration membrane at a pH in the range of 5.5 to 8 and the diafiltration in step (g) is carried out on 10 kDa ultrafiltration membrane at a pH in the range of 6.3 to 7.3.

28. The method as claimed in claim 1, wherein step (e) is carried out in the presence of agitation.

29. The method as claimed in claim 1, wherein step (e) is carried out in the absence of agitation.

30. The method as claimed in claim 1, wherein a salt selected from the group consisting of KCl, NaCl, CaCl.sub.2, MgCl.sub.2 and NH.sub.4Cl is added prior to the treatment with an oxidizing agent in step (e), and wherein the final concentration of the salt is in the range of 0.5 M to 10 M.

31. (canceled)

32. (canceled)

33. The method as claimed in claim 1, wherein the supernatant of step (f) is incubated at a temperature in the range of 4° C. to 80° C., for a time period in the range of 2 hours to 15 hours.

34. The method as claimed in claim 33, wherein the incubation is carried out in the absence of agitation.

35. The method as claimed in claim 33, wherein the incubation is carried out in the presence of agitation.

36. The method as claimed in claim 1, wherein the method does not utilize any chromatography, alcohol, detergent and enzymes.

37. The method as claimed in claim 1, wherein the method utilizes one or more of chromatography, alcohol, detergent and enzymes.

38. The method as claimed in claim 37, wherein the method comprises an additional step of chromatography to obtain the purified and sized polysaccharide, wherein the chromatography is selected from the group consisting of ion-exchange (cationic or anionic), affinity chromatography, hydrophilic-interaction, hydrophobic-interaction, size-exclusion, hydroxyapatite, 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.

39. The method as claimed in claim 37, wherein the method comprises an additional step of treatment with one or more chemical/biological reagents at any stage of the method, 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 α-olefin sulfonates, sodium alkyl sulfonates, α-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, RNase, Benzonase, Mutanolysin/lysozyme, beta.-D-N-acetyl glucosaminidase, and Proteinase K.

40. A bacterial polysaccharide obtained by the method as claimed in claim 1 having the molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), the polydispersity in the range of 1 to 5, the hexosamine content between 10% and 55%, the pyruvate content greater than 10%, the protein content less than 2%, the nucleic acid content less than 2%, the immunoreactivity in the range of 40% to 95%, and the percentage % yield of polysaccharide in the range of 60% to 95%.

41. The bacterial polysaccharide as claimed in claim 40, wherein the molecular size is in the range of 100 kDa to 300 kDa (SEC-HPLC).

42. The bacterial polysaccharide as claimed in claim 40, wherein the polysaccharide is used for preparation of plain polysaccharide.

43. The bacterial polysaccharide as claimed in claim 40, polysaccharide is used for preparation of polysaccharide-protein conjugate vaccine.

44. An immunogenic composition comprising Streptococcus pneumoniae polysaccharides, wherein the polysaccharide is obtained by the method as claimed in claim 1 having the molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), the polydispersity in the range of 1 to 5, the hexosamine content between 10% and 55%, the pyruvate content greater than 10%, the protein content less than 2%, the nucleic acid content less than 2%, the immunoreactivity in the range of 40% to 95%, and the percentage yield of polysaccharide in the range of 60% to 95%, and wherein the polysaccharides are derived from Streptococcus pneumoniae serotypes selected from the group consisting of 1, 2, 3, 4, 5, 6, 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, 18, 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.

45. The immunogenic composition as claimed in claim 44, wherein the polysaccharides are conjugated to a peptide, a protein or an organic polymer, preferably a carrier protein selected from CRM.sub.197, 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.

46. The immunogenic composition as claimed in claim 44, wherein the polysaccharides are conjugated to the carrier protein using one of cyanylation chemistry, CNBr chemistry, reductive amination chemistry, and carbodiimide chemistry.

47. The immunogenic composition as claimed in claim 46, 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.

48. The immunogenic composition as claimed in claim 46, wherein the conjugates have free polysaccharide less than 3.5% and free protein less than 1.8%.

49. The immunogenic composition as claimed in claim 46, wherein the polysaccharide is 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.

50. The immunogenic composition as claimed in claim 46, wherein the polysaccharide is conjugated to the carrier protein in the absence of a linker.

51. The immunogenic composition as claimed in claim 46, wherein serotype 4 has a pyruvate content of at least 16% and hexosamine content in the range of 30% to 45%.

52. The immunogenic composition as claimed in claim 46 comprises pharmaceutically acceptable excipients selected from surfactants, stabilizers, buffers, diluents, adjuvants, preservatives and solvents.

53. The immunogenic composition as claimed in claim 52 wherein the preservative is selected from the group consisting 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.

54. The immunogenic composition as claimed in claim 36, wherein the composition is one of a 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.

55. An immunogenic composition comprising 10 distinct Streptococcus pneumoniae polysaccharide—protein conjugates wherein the polysaccharides are obtained by the method as claimed in claim 1 having the molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), the polydispersity in the range of 1 to 5, the protein content less than 2%, the nucleic acid content less than 2%, the immunoreactivity in the range of 40% to 95%, and the percentage yield of polysaccharide in the range of 60% to 95%, wherein the polysaccharides are derived from serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F, and wherein all the polysaccharides are conjugated to CRM.sub.197 as the carrier protein.

56. An immunogenic composition comprising 17 distinct Streptococcus pneumoniae polysaccharide—protein conjugates wherein the polysaccharides are obtained by the method as claimed in claim 1 having the molecular size in the range of 5 kDa to 900 kDa (SEC-HPLC), the polydispersity in the range of 1 to 5, the protein content less than 3%, the nucleic acid content less than 2%, the immunoreactivity in the range of 40% to 95%, and the percentage yield of polysaccharide in the range of 60% to 95%, 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, and wherein serotype 3 is conjugated to CRM.sub.197, serotype 18C is conjugated to CRM.sub.197, serotype 4 is conjugated to DT, serotype 15B is conjugated to either TT, DT or CRM.sub.197, and serotype 22F is conjugated to TT.

Description

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING

[0076] The present disclosure will now be described with the help of the accompanying drawing, in which:

[0077] FIG. 1 illustrates the literature .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 4;

[0078] FIG. 2 illustrates the .sup.1H NMR spectrum purified S. pneumoniae polysaccharide for serotype 4 obtained by the method of present disclosure;

[0079] FIG. 3 illustrates the literature spectrum of purified S. pneumoniae polysaccharide for serotype 1; and

[0080] FIG. 4 illustrates the .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 1 obtained by the method of present disclosure.

DESCRIPTION

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The present disclosure envisages a simple and cost effective method for obtaining size reduced and 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 proteins.

[0086] In an aspect of the present disclosure, there is provided a method for obtaining purified bacterial polysaccharides. The method comprises removal of impurities such as proteins and nucleic acid from the crude polysaccharides and sizing of the polysaccharides to obtain purified bacterial polysaccharides having the desired molecular size.

[0087] In an embodiment of the present disclosure, the method comprises the following steps: [0088] providing a fermentation harvest comprising bacterial cells expressing a polysaccharide; [0089] inactivating the bacterial cells in the fermentation harvest by inactivating agent to the fermentation harvest; [0090] centrifuging the fermentation harvest comprising the inactivated bacterial cells to separate the cell free supernatant; [0091] concentrating and filtering the cell free supernatant; and [0092] treating the concentrated and filtered cell free supernatant with an oxidizing agent; [0093] centrifuging the above mixture to collect the supernatant; and [0094] diafiltering and concentrating the supernatant to obtain the sized and purified polysaccharide having molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), polydispersity in the range of 1 to 5, hexosamine content between 10% and 55%, pyruvate content greater than 10%, protein content less than 2%, nucleic acid content less than 2%, immunoreactivity in the range of 40% to 95%, and % yield of polysaccharide in the range of 60% to 95%.

[0095] 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. such as Shigella sonnei, Shigella flexneri, Shigella dysenteriae; Shigella boydii; E. coli; Neisseria meningitidis (serotypes such as A, B, C, D, 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 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.

[0096] 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, 6, 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, 18, 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.

[0097] The bacteria used in the present disclosure can be sourced from known collection centres, such as CDC, Atlanta USA; CBER/FDA, USA; ATCC, USA; NIH, USA; NIAID, USA; and PHE, UK.

[0098] The bacterial culture is subjected to fermentation to obtain a fermentation harvest comprising bacterial cells expressing a polysaccharide. The fermentation is carried out using optimum fermentation parameters, such as medium, pH, temperature, oxygen amount and time period. The fermentation is typically carried out at a pH in the range of 3 to 9, preferably at a pH of 5 to 8.

[0099] The bacterial cells in the fermentation harvest are then inactivated. The bacterial cells may be inactivated using known techniques, to release the polysaccharide. In accordance with the embodiments of the present disclosure, inactivation may be carried out using an inactivating agent including detergents (such as deoxycholate sodium (DOC)), enzymes (such as lysozymes), mechanical/physical means (such as sonication), and the like. The fermentation harvest containing the inactivated/lysed bacterial cells may be then centrifuged and filtered.

[0100] Centrifugation helps in separation and removal of undesired cell debris, colloids and large aggregates thereby reducing burden on downstream processing. Centrifugation may be carried out 10000 g to 20000 g, 10° C. to 20° C. to obtain cell free supernatant having pH in the range of 5.0 to 7.0, preferably 5.8 to 6.8. In an embodiment, the centrifugation may be carried out for a time period in the range of 20 minutes to 180 minutes.

[0101] In a preferred embodiment, the inactivating agent is deoxycholate sodium (DOC).

[0102] The cell free supernatant is typically subjected to diafiltration and concentration to obtain crude polysaccharide. In one embodiment, the diafiltration and concentration of harvested supernatant is carried out using 100 kDa cutoff ultrafiltration membrane to obtain crude polysaccharide (pH 5.8 to 7.4). 10× diafiltration (DF) against 150 mM NaCl solution and followed by ˜20× DF against WFI to obtain crude polysaccharide having molecular weight in the range of 600 kDa to 1000 kDa.

[0103] The crude/native polysaccharide is then treated with an oxidizing agent to obtain purified and sized polysaccharide having reduced molecular weight.

[0104] As used in the present disclosure, the term ‘sizing/fragmentation/de-polymerization’ refers to reducing the size of the bacterial polysaccharide in accordance using the process described herein to obtain a polysaccharide having reduced/desired molecular size as compared to the original polysaccharide.

[0105] The inventors of the present disclosure have found that depolymerised/sized polysaccharides have following advantages over plain polysaccharides when used to prepare the conjugates: (a) the conjugates prepared from using depolymerized polysaccharides may be inherently more immunogenic than the corresponding conjugates prepared from full length polysaccharides; and (b) reactions used to prepare these conjugate vaccines can offer a higher degree of control, as well as more versatility in process design, when using depolymerized polysaccharide chains versus full length polysaccharide chains.

[0106] The inventors of the present disclosure have further found that it is necessary to reduce the molecular size of the polysaccharide prior to the coupling reaction in order to perform end-group conjugation with high yields. Size reduction of polysaccharides decreases the viscosity of the solution and increases the number of reactive end groups, both factors contribute to an increased frequency of covalent bond formation. Furthermore the optimal immunogenicity of polysaccharide components of conjugate vaccines is said to be related to their molecular size.

[0107] In an embodiment of the present disclosure, the oxidizing agent is a peroxide selected from the group including, but not limited to, peroxy acids (such as peracetic acid), H.sub.2O.sub.2, metal peroxides (such as barium peroxide, sodium peroxide), and organic peroxides (such as tert-butylhydroperoxide).

[0108] In a preferred embodiment of the present disclosure, the peroxide is H.sub.2O.sub.2.

[0109] The step involves adding a salt to the crude polysaccharide, followed by addition of the oxidizing agent.

[0110] Typically, the salt can include, but is not limited to, KCl, NaCl, CaCl.sub.2, MgCl.sub.2 and NH.sub.4O. The final concentration of the salt, can be in the range of 0.5 M to 10 M, preferably 0.5 NI to 7 M, depending on the salt. The salt is allowed to completely dissolve prior to the addition of the oxidizing agent.

[0111] In an embodiment of the present, disclosure, oxidizing agent is then added and the mixture is incubated at a pre-determined temperature for a pre-determined time period to obtain purified polysaccharide having reduced molecular weight.

[0112] Typically, the oxidizing agent is added such that the final concentration is in the range 0.5% to 50%, 2% to 50%, 2% to 20%, 5% to 10%, preferably 7.5%.

[0113] The mixture containing the oxidizing agent can be incubated at a temperature in the range of 4° C. to 80° C., 4° C. to 70° C., 10° C. to 40° C., 15° C. to 35° C., 25° C. to 35° C., 28° C. to 32° C., preferably 30° C.

[0114] The mixture containing oxidizing agent can be incubated for a time period in the range of 2 hours to 15 hours.

[0115] In an embodiment of the present disclosure, the incubation is carried out in the presence of a reagent selected from the group comprising buffers, solvents, metal salts, organic acids and mineral acids. The buffer is selected from the group consisting of TRIS, sodium phosphate, sodium acetate; the organic acid is selected from the group consisting of ascorbic acid, acetic acid, citric acid; and the mineral acid is selected from the group consisting of HCl, H.sub.2SO.sub.4, TFA, and HNO.sub.3.

[0116] In an embodiment of the present disclosure, the incubation is carried out in the absence of agitation.

[0117] In another embodiment of the present disclosure, the incubation is carried out in the presence of agitation.

[0118] The mixture is then centrifuged to obtain clear supernatant and the pellet is discarded. In an embodiment, centrifugation is carried out using a Relative Centrifugal Force (RCF) of 10000 g to 20000 g at a temperature in the range of 2° C. to 20° C. for a time period in the range of 30 minutes to 120 minutes.

[0119] The clear supernatant may be further incubated at a temperature in the range of 4° C. to 80° C., for a time period in the range of 2 hours to 15 hours.

[0120] In an embodiment of the present disclosure, the incubation is carried out in the absence of agitation.

[0121] In another embodiment of the present disclosure, the incubation is carried out in the presence of agitation.

[0122] The sized polysaccharide having reduced impurities is typically subjected to diafiltration and concentration to obtain purified and sized polysaccharide. In an embodiment, diafiltration and concentration is carried out using 10 kDa cutoff ultrafiltration membrane against WFI to obtain purified sized polysaccharide (pH 6.8±0.5).

[0123] 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 purified and sized bacterial polysaccharide obtained by the method of the present disclosure is in the range of 5 kDa to 900 kDa, 50 kDa to 600 kDa, preferably 50 kDa to 300 kDa, still preferably 50 kDa to 250 kDa. In an exemplary embodiment of the present disclosure, the average molecular size of the purified bacterial polysaccharide is in the range of 100 kDa to 200 kDa.

[0124] 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.

[0125] The purified and sized bacterial polysaccharide obtained by the method of the present disclosure has optimal characteristics:

[0126] molecular weight in the range of 5 kDa to 900 kDa (as determined by SEC-HPLC); [0127] polydispersity in the range of 1 to 5, preferably 1 to 3; [0128] hexosamine content between 10% and 55%, 10% to 40%, preferably between 30% and 45%; [0129] immunoreactivity in the range of 40% to 95%, preferably between 45% and 90%; [0130] protein content less than 2%; [0131] nucleic acid content less than 2%; [0132] pyruvate content greater than 10%, preferably 10% to 20%; and [0133] % yield of polysaccharide in the range of 60% to 95%, preferably between 70% and 90%.

[0134] Typically, the recovery of purified polysaccharides using the method of the present disclosure is more than 70% compared to about 55% recovery of polysaccharides reported in literature.

[0135] 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 0.2% and nucleic acids to less than 0.3% from 15% and 25%, respectively.

[0136] The method of the present disclosure using an oxidizing agent for simultaneous sizing and removal of impurities makes it possible to obtain purified and sized polysaccharides having optimal pyruvate content and hence maintains CPS integrity, confers enhanced stability and immunogenicity to the polysaccharide and conjugate obtained thereof. The method of the present disclosure is also devoid of costly chromatography steps that makes it economical for large scale manufacturing.

[0137] The present disclosure provides low polydispersity polysaccharide, wherein such low polydispersity polysaccharide (CPS) gives conjugate with narrow distribution of size. This helps to have better conjugation process control, higher recoveries when concentrated on a membrane as well as ease in purification of crude polysaccharide protein conjugate. Most importantly by applicant's method the polysaccharide antigen remains structurally intact, i.e. the epitopic conformation is retained which ensures that resultant polysaccharide-protein conjugate shows optimal immunogenicity

[0138] In another embodiment of the present disclosure, the crude bacterial polysaccharide may be subjected to additional purification steps before treatment with the oxidizing agent.

[0139] In an embodiment of the present disclosure, the method may comprise any subset or all of the following steps before treatment with the oxidizing agent: [0140] treating the crude/native polysaccharide with an enzyme to remove impurities; [0141] acid precipitation of impurities; [0142] separation of the precipitation, followed by centrifugation to obtain a supernatant; [0143] subjecting the supernatant to further purification using a chromatographic method; and [0144] diafiltration and concentration of the chromatographic flowthrough to obtain purified native polysaccharide.

[0145] In an embodiment of the present disclosure, the method does not utilize any additional step of chromatography, alcohol, detergent and enzymes.

[0146] In another embodiment of the present disclosure, the method utilizes one or more additional steps of chromatography, alcohol, detergent and enzymes.

[0147] The crude polysaccharide is treated with an enzyme, such as protease, benzonase to remove impurities. The enzyme treatment can be carried out at a pH in the range of 7.0 to 8.5, in the presence of an acid and a salt. The concentration of the enzyme can be in the range of 2 IU/ml to 20 IU/ml. In an embodiment, the acid may be Tris HCl having a concentration in the range of 5 mM to 50 mM. The salt is selected from NaCl, CaCl.sub.2, MgCl.sub.2, and has a concentration in the range of 0.5 mM to 1 mM. The enzyme treatment can be carried at a temperature in the range of 36° C. to 38° C. and for a time period of 5 hours to 15 hours.

[0148] Thereafter, the mixture is precipitated using 10% to 30% acid. In an embodiment, the acid is a mineral acid, such as HCl, HNO.sub.3, and H.sub.2SO.sub.4. The pH is adjusted to 5 to 8 using the acid to precipitate the impurities.

[0149] Thereafter, the precipitated impurities are separated by centrifugation and the supernatant comprising the polysaccharide is collected for further processing.

[0150] The supernatant comprising the polysaccharide is subjected to additional purification using chromatographic techniques. In an embodiment, the supernatant comprising the polysaccharide is subjected to Hydrophobic Interaction Chromatography (HIC), Ion Exchange Chromatography. In a preferred embodiment, the HIC resin is Octyl Sepharose 4FF having pH in the range of 6 to 7.5.

[0151] The HIC flowthrough is diafiltered and concentrated. In an embodiment, the HIC flowthrough is diafiltered and concentrated using 100 kDa cutoff ultrafiltration membrane to obtain purified native polysaccharide (pH 6.5±0.5), which is then subjected to treatment with an oxidizing agent as described previously.

[0152] In yet another embodiment, the method of the present disclosure may include at least one of the following at any stage of the method of the present invention to obtain the sized and purified bacterial polysaccharide: [0153] treatment with reagents, such as cetyl trimethyl ammonium bromide (CTAB), hexadimethrine bromide and myristyltrimethylammonium, triton, acetate (such as ethyl acetate, sodium acetate, barium acetate, calcium acetate, ammonium acetate, cupric acetate), sodium carbonate, zinc, enzymes (protease, endonuclease, DNase, RNase, benzonase, mutanolysin/lysozyme, beta.-D-N-acetyl glucosaminidase, and proteinase K), ethanol, acetone, CaCl.sub.2, sodium dodecyl sulfate (SDS), polysorbate 20, polysorbate 80, sodium sarcosine, NaCl, urea, formaldehyde, ammonium chloride, ethylenediaminetetraacetic acid (EDTA), ammonium sulfate, mineral acid (such as HCl, H.sub.2SO.sub.4, HNO.sub.3, TCA), organic acid (such as formic acid, acetic acid, propionic acid, and oxalic acid), iso propanol, metal cations (such as Ca, Mg, Mn, Fe, Cu, Co, Mo), butanol, toluene and chloroform, alkali (such as sodium hydroxide, potassium hydroxide, sodium carbonate, hydroxyl amine, triethyl amine and lithium hydroxide), 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 α-olefin sulfonates, sodium alkyl sulfonates, α-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and sodium alkyl glyceryl ether sulfonates; [0154] chromatographic steps, such as affinity chromatography, hydrophilic-interaction, hydrophobic-interaction, size-exclusion, ion exchange chromatography (cationic or anionic), Cibacron Blue pseudo affinity sorbent, mixed mode chromatography sorbent, membrane chromatography, monolith chromatography device, adsorbent chromatography, lectin agarose column, hydroxyapatite chromatography, Amberlite column, gel-permeation chromatography, Capto Adhere, TOYOPEARL MX-Trp-650M, Cellufine MAX AminoButyl, adsorbent chromatography; and [0155] filtration methods such as depth filtration, carbon filtration, ultrafiltration, gel filtration.

[0156] The bacterial polysaccharides obtained by the method of the present disclosure may be used as antigens, or may be used for production of antibodies, and vaccines. The polysaccharides may be used alone or may be conjugated to carrier protein. Still further, the polysaccharides exhibit enhanced immunogenicity and stability when compared with polysaccharides prepared using conventional methods.

[0157] The present disclosure envisages a bacterial polysaccharide having molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), polydispersity in the range of 1 to 5, hexosamine content between 10% and 55%, pyruvate content greater than 10%, protein content less than 2%, nucleic acid content less than 2%, immunoreactivity in the range of 40% to 95%, and % yield of polysaccharide in the range of 60% to 95%.

[0158] In an embodiment of the present disclosure, the molecular size of the bacterial polysaccharide is in the range of 100 kDa to 300 kDa (SEC-HPLC).

[0159] In yet another embodiment, the bacterial polysaccharide is used for preparation of polysaccharide-protein conjugate vaccine.

[0160] In an embodiment of the present disclosure, S. pneumoniae serotype 4 polysaccharide obtained by the method of the present disclosure has a pyruvate content of at least 16% and hexosamine content in the range of 30% to 45%.

[0161] In accordance with the embodiments of the present disclosure, the polysaccharides obtained by the method of the present disclosure may be conjugated to a peptide, a protein or an organic polymer, preferably a carrier protein selected from the group consisting of Tetanus toxoid (TT), CRM.sub.197 (Cross Reactive Material 197, a genetically detoxified form of diphtheria toxoid), 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 (HSA), 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.

[0162] Typically, the polysaccharide of the present disclosure may be conjugated to the carrier protein using an appropriate conjugation chemistry, including, but not limited to, cyanylation chemistry, CNBr chemistry, reductive amination chemistry, and carbodiimide chemistry. The cyanylation agent may include, but is not limited to, 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. Typically the conjugates prepared using the polysaccharides obtained using the method of the present disclosure have free polysaccharide less than 3.5% and free protein less than 1.8%.

[0163] In one embodiment of the present disclosure, the polysaccharide is conjugated to the carrier protein in the presence of a linker. The linker may be selected from the group comprising hexanediamine, ethylene diamine, hydrazine, adipic dihydrazide, and 1,6-diaminooxyhexane.

[0164] In another embodiment of the present disclosure, the polysaccharide is conjugated to the carrier protein in the absence of a linker.

[0165] Purified and sized polysaccharide of the present disclosure may also be used as a plain polysaccharide vaccine.

[0166] In a preferred embodiment, vaccine compositions are “Monovalent” or “Multivalent” polysaccharide-protein conjugate vaccine compositions prepared using the polysaccharides of the present disclosure are single dose compositions free of preservatives.

[0167] Conjugates obtained by the present disclosure may be further combined with heat shock proteins (HSPs), and whole cell for preparing S. pneumoniae vaccine composition.

[0168] The present disclosure envisages immunogenic compositions comprising 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.

[0169] The present disclosure envisages an immunogenic composition comprising Streptococcus pneumoniae polysaccharides, wherein the polysaccharide is obtained by the method of the present disclosure. The polysaccharides have a molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), polydispersity in the range of 1 to 5, hexosamine content between 10% and 55%, pyruvate content greater than 10%, protein content less than 2%, nucleic acid content less than 2%, immunoreactivity in the range of 40% to 95%, and % yield of polysaccharide in the range of 60% to 95% and the polysaccharides are derived from Streptococcus pneumoniae serotypes selected from the group consisting of 1, 2, 3, 4, 5, 6, 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, 18, 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.

[0170] In an embodiment of the present disclosure, there is provided an immunogenic composition comprising 10 distinct Streptococcus pneumoniae polysaccharide—protein conjugates. The polysaccharides are obtained by the method of the present disclosure having molecular weight in the range of 5 kDa to 900 kDa (SEC-HPLC), polydispersity in the range of 1 to 5, protein content less than 2%, nucleic acid content less than 2%, immunoreactivity in the range of 40% to 95%, and % yield of polysaccharide in the range of 60% to 95%. The polysaccharides are derived from Streptococcus pneumoniae serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F; and all the polysaccharides are conjugated to CRM.sub.197 as the carrier protein.

[0171] In another embodiment of the present disclosure, there is provided an immunogenic composition comprising 17 distinct Streptococcus pneumoniae polysaccharide—protein conjugates. The polysaccharides are obtained by the method of the present disclosure having molecular size in the range of 5 kDa to 900 kDa (SEC-HPLC), polydispersity in the range of 1 to 5, protein content less than 3%, nucleic acid content less than 2%, immunoreactivity in the range of 40% to 95%, and % yield of polysaccharide in the range of 60% to 95%. The polysaccharides are derived from Streptococcus pneumoniae 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 CRM.sub.197, serotype 18C is conjugated to CRM.sub.197, serotype 4 is conjugated to DT, serotype 15B is conjugated either to TT, DT or CRM.sub.197, and serotype 22F is conjugated to TT.

[0172] Typically, the immunogenic composition comprises pharmaceutically acceptable excipients selected from surfactants, stabilizers, buffers, diluents, adjuvants, preservatives and solvents.

[0173] In another embodiment, 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.

[0174] The method of the present disclosure is cost effective with high recovery of sized and purified polysaccharides, and can be performed very easily.

[0175] 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

[0176] The Streptococcus pneumoniae serotypes 4 and 1 were sourced from Centers for Disease Control and Prevention (CDC), Atlanta, USA.

[0177] U.S. Pat. No. 9,249,439 is being incorporated with reference to the fermentation processes utilized for all S. pneumoniae serotypes

Example-1: Obtaining Fragmented and Purified Polysaccharide from Streptococcus Pneumoniae Serotype 4 in Accordance with the Method of the Present Disclosure

[0178] The method for obtaining fragmented and purified polysaccharide in accordance with the present disclosure comprised the following steps: [0179] Fermentation process was carried out at pH 7.1±0.5. [0180] The fermentation harvest comprising S. pneumoniae serotype 4 cells were inactivated using DOC [12% stock solution was used, which was added in fermenter to make the final concentration of 2.5%], followed by centrifugation (at 15000 g, 15° C., 60 minutes) to get cell free supernatant harvest (pH 5.8 to 6.8). [0181] The cell free supernatant harvest was diafiltered and concentrated using 100 kDa cutoff ultrafiltration membrane to obtain crude polysaccharide (pH 5.8 to 7.4). 10× DF against 150 mM NaCl solution and followed by ˜20× DF against WFI. Molecular weight of crude polysaccharide is ˜1000 kDa to 600 kDa. [0182] H.sub.2O.sub.2 treatment was carried out for size reduction of crude polysaccharide: in crude native polysaccharide by adding 2M equivalent solid NaCl and dissolving it completely, followed by addition of 25% (v/v) equivalent H.sub.2O.sub.2 (stock concentration 30%) such that the final concentration of H.sub.2O.sub.2. is ˜7.5%, (pH 6.2±0.5). [0183] The mixture obtained in the above step is incubated at 30° C.±1.0° C., without agitation. [0184] After incubation, the reaction mixture was centrifuged (RCF=15000 g, ˜60 minutes, 10° C.) to get precipitate free clear supernatant. [0185] After centrifugation, the supernatant was collected and the precipitated pellet was discarded. [0186] The supernatant is further incubated at 30° C.±1.0° C., without agitation. [0187] After 2 hours to 5 hours OR on achieving the desired molecular weight (i.e. 150±50 kDa), the reaction mixture was diafiltered and concentrated using 10 kDa cutoff ultrafiltration membrane to obtain fragmented/sized and purified polysaccharide (pH 6.8±0.5).

[0188] The sized and purified polysaccharide was characterized and the results obtained are illustrated in Table-1:

TABLE-US-00001 TABLE 1 Experimental data specifications for Serotype 4 sized and S. No. purified polysaccharide 1) Mw by SEC-HPLC (kDa) 700 ± 50 kDa to 150 ± 50 kDa 2) Polydispersity 2.0 ± 0.5  3) Pyruvate content 16 ± 2% 4) Hexosamine 40 ± 2%

[0189] Further, the experimental data specification for Serotype 4 conjugate is provided in Table-la:

TABLE-US-00002 TABLE 1a S. No. Experimental data specifications for Serotype 4 conjugate 1) Mw by SEC-HPLC (kDa) 850 ± 60 kDa 2) Polydispersity 3.1 ± 0.2  .sup.  3) Pyruvate content 16 ± 2% .sup.  4) Conjugate stability Stable 5) Conjugate immunogenicity Passes 6) Free Ps (%) <3.15 7) Free protein (%) <1.8 

[0190] Similarly purified polysaccharide from serotype 1 was also obtained using the method of the present disclosure.

[0191] NMR analysis of the purified polysaccharides was carried out and the results obtained are illustrated in FIGS. 1-4.

[0192] Figure-1 illustrates the literature .sup.1H NMR spectrum of purified S. pneumoniae polysaccharide for serotype 4, and Figure-2 illustrates the .sup.1H NMR spectrum purified S. pneumoniae polysaccharide for serotype 4 obtained by the method of present disclosure.

[0193] Interpretation of NMR Analysis of Serotype 4:

[0194] The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharide. A portion of the anomeric region of each spectrum (4.5 to 6.0 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units.

[0195] Anomeric Center:

[0196] Typically the chemical shifts for anomeric protons found further downfield (higher ppm) on the NMR spectrum than other ring protons, generally 4-6 ppm. Because of their distinctive shifts, these signals are generally the most diagnostic component of the .sup.1H NMR spectrum.

[0197] Identity of Serotype 4 Polysaccharide by 1D Proton NMR

[0198] Pneumococcal polysaccharide of serotype 4 is a tetra-saccharide repeating unit having 4 anomeric signals or protons. 1D Proton NMR spectra of ATCC 4 pneumococcal polysaccharide (PnPs) (Figure-1) was compared to PnPs NMR spectrum obtained for serotype 4 polysaccharide obtained by method of present disclosure (Figure-2). Spectra looks similar to each other with respect to anomeric region (4.5-6.0 ppm), N-acetyl Fucose at 1.25 ppm, Pyruvate at 1.5-1.6 ppm, N-acetyl at 1.9-2.1 ppm and -PCho peak (3.23 ppm) which represents the presence of C-Ps in PnPs preparation.

[0199] Therefore, identity of PnPs for serotype 4 obtained by the method of the present disclosure was confirmed by comparing the ATCC PnPs as reference standard by proton NMR with peaks identification.

[0200] Interpretation of NMR Analysis of Serotype 1:

[0201] The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharide. A portion of the anomeric region of each spectrum (4.5 to 6.0 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units.

[0202] Anomeric Center:

[0203] Typically the chemical shifts for anomeric protons found further downfield (higher ppm) on the NMR spectrum than other ring protons, generally 4-6 ppm. Because of their distinctive shifts, these signals are generally the most diagnostic component of the .sup.1H NMR spectrum.

[0204] Identity of Serotype 1 Polysaccharide by 1D Proton NMR

[0205] 1D Proton NMR spectra of ATCC 1 PnPs were compared to PnPs for serotype 1 polysaccharide obtained by the method of present disclosure. Spectra looks similar to each other with respect to anomeric region (4.5-6.0 ppm), N-acetyl and O-acetyl signal at 1.9-2.1 ppm and -PCho peak (3.23 ppm) which represents the presence of C-Ps in PnPs preparation.

[0206] Therefore, identity of PnPs for serotype 1 obtained by the method of the present disclosure was confirmed by comparing the ATCC PnPs as reference standard by proton NMR with peaks identification.

[0207] Pneumococcal polysaccharide of serotype 1 is a tri-saccharide repeating unit having 3 anomeric signals or protons. 1D Proton NMR spectra of ATCC 1 PnPs (Figure-3) was compared to PnPs NMR spectrum obtained for serotype 1 polysaccharide by method of the present disclosure (Figure-4). Spectra looks similar to each other with respect to anomeric region (4.5-6.0 ppm), N-acetyl and O-acetyl signal at 1.9-2.1 ppm and -PCho peak (3.23 ppm) which represents the presence of C-Ps in PnPs preparation

[0208] Therefore, identity of PnPs obtained by method of present disclosure for serotype 1 was confirmed by comparing the ATCC PnPs as reference standard by proton NMR with peaks identification.

[0209] It is seen from Table-1 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.

[0210] Similar results were obtained for S. pneumoniae serotype 1 purified and sized polysaccharide using the method of the present disclosure. Experiments were also carried out using different concentrations of H.sub.2O.sub.2. It was seen that at concentrations of 0.5%, 1% and 3% only moderate changes in molecular impurity (protein and nucleic acid) profile was observed which does not meet the quality criteria specified by WHO-TRS/pharmacopeia. Also the rate of depolymerization/sizing of polysaccharide is slow as compared to optimal H.sub.2O.sub.2 concentration of 7.5% in accordance with the present disclosure. At concentration 5% H.sub.2O.sub.2, similar result as for 7% was observed. On the other hand, at concentrations of 10%, 30-50%, 35% H.sub.2O.sub.2, the rate of polysaccharide depolymerization increases, however very low recovery of polysaccharide is observed.

[0211] Experiments were carried out using different temperature during incubation after addition of H.sub.2O.sub.2. It was seen that at 15° C. the rate of polysaccharide depolymerization is slow but the results are similar that of 30° C. At 45° C., 50° C., 60° C., 70° C., 80° C. the rate of polysaccharide depolymerization increases as temperature increases, however, very low recovery of polysaccharide was observed.

Example-2: Obtaining Fragmented and Purified Polysaccharide from Streptococcus Pneumoniae Serotype 4 Using Benzonase+Hydrophobic Interaction Chromatography (HIC)

[0212] The method for obtaining fragmented and purified polysaccharide using benzonase+HIC comprised the following steps: [0213] Fermentation process was carried out at pH 7.1±0.5. [0214] The fermentation harvest comprising S. pneumoniae serotype 4 cells were inactivated using DOC [12% stock solution was used, which was added in fermenter to make the final concentration of 2.5%], followed by centrifugation (at 15000 g, 15° C., 60 minutes) to get cell free supernatant harvest (pH 5.8 to 6.8). [0215] The cell free supernatant harvest was diafiltered and concentrated using 100 kDa cutoff ultrafiltration membrane to obtain crude polysaccharide (pH 5.8 to 7.4). 10× DF against 150 mM NaCl solution and followed by ˜20× DF against WFI. Molecular weight of crude polysaccharide was ˜1000 kDa to 600 kDa. [0216] Benzonase treatment to crude polysaccharide (pH 8.0±0.2): Reaction mixture consisting crude pneumococcal polysaccharide+Benzonase at concentration of 10 IU/mL+Tris HCl at concentration of 20 mM+MgCl.sub.2 at concentration of 2 mM for 10±2 hours at 37° C. [0217] Impurities were precipitated by adjusting the pH 5.8±0.2 using 20% HCl. [0218] The precipitate was separated by centrifugation and supernatant was collected. [0219] The supernatant was further purified using Hydrophobic interaction chromatography (HIC resin is Octyl sepharose 4FF) (pH 6.8±0.5). [0220] The HIC flowthrough was diafiltered and concentrated using 100 kDa cutoff ultrafiltration membrane to obtain purified native polysaccharide (pH 6.5±0.5). [0221] H.sub.2O.sub.2 treatment was carried out for size reduction of crude polysaccharide: in crude native polysaccharide by adding 2M equivalent solid NaCl and dissolving it completely, followed by addition of 25% (v/v) equivalent H.sub.2O.sub.2 (stock concentration 30%) such that the final concentration of H.sub.2O.sub.2. is ˜7.5%, (pH 6.2±0.5). [0222] The mixture obtained in the above step was incubated at 30° C.±1.0° C., without agitation. [0223] After incubation, the reaction mixture was centrifuged (RCF=15000 g, —60 minutes, 10° C.) to get precipitate free clear supernatant. [0224] After centrifugation, the supernatant was collected and the precipitated pellet was discarded. [0225] The supernatant was further incubated at 30° C.±1.0° C., without agitation. [0226] After 2 hours to 5 hours OR on achieving the desired molecular weight (i.e. 150±50 kDa), the reaction mixture was diafiltered and concentrated using 10 kDa cutoff ultrafiltration membrane to obtain fragmented/sized and purified polysaccharide (pH 6.8±0.5).

[0227] Experiments were carried out by varying the concentration of benzonase, it was observed that benzonase 5 to 10 IU and higher concentrations were effective in removing the DNA impurity. Further, it was found that MgCl.sub.2 was essential for activity of benzonase, in absence of MgCl.sub.2 the enzymatic activity was not observed but in presence of MgCl.sub.2 the enzymatic activity was found to be satisfactory and resulted in efficient removal of DNA impurity. The effect of different incubation duration [2 hours to 10 hours] on DNA impurity removal was studies. It was observed from the results obtained that on 10 hours incubation, DNA impurity was not detected; whereas below 9 hours DNA impurity was detected. Hence, it was concluded that, as the duration of incubation increases, DNA impurity concentration decreases.

TECHNICAL ADVANTAGES

[0228] 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: [0229] a method for purification of bacterial polysaccharides with high recovery and low impurity content; [0230] obtaining purified and sized polysaccharides having; [0231] molecular weight in the range of 5 kDa to 900 kDa (as determined by SEC-HPLC); [0232] polydispersity in the range of 1 to 5, preferably 1 to 3; [0233] hexosamine content between 10% and 55%, 10% to 40%, preferably between 30% and 45%; [0234] immunoreactivity in the range of 40% to 95%, preferably 45% to 90%; [0235] protein content less than 2%; [0236] nucleic acid content less than 2%; [0237] pyruvate content greater than 10%, preferably 10% to 20%; and [0238] % yield of polysaccharide in the range of 60% to 95%, preferably 70% and 90%. [0239] obtaining purified bacterial polysaccharide having high polysaccharide recovery (at least 60%) and low impurity content (proteins 0.2% and nucleic acids 0.3%); and [0240] simple, rapid and cost effective method for purification of bacterial polysaccharides that does not utilize phenol, triton, enzymes, CTAB, activated carbon, chromatography (such as HIC), ammonium sulphate or ethanol.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.