Method for removal of impurities from bacterial capsular polysaccharide based preparations

Abstract

The present invention relates to an improved process for purification of bacterial capsular polysaccharides, more specifically capsular polysaccharides of gram negative bacteria. The process comprises of concentration and dia filtration of harvest, treatment with anionic detergent and strong alkali followed by centrifugation, diafiltration and cationic detergent based precipitation of bacterial polysaccharides. The process results in significant reduction of endotoxin, protein and nucleic acid impurities thereby providing higher recovery of capsular polysaccharide with the desired O-acetyl levels. Said process is scalable, non-enzymatic, and employs fewer purification steps.

Claims

1. A method for isolation of polysaccharide from Neisseria meningitidis selected from serotype A and serotype X, said method comprising the following steps: a) adding a detergent comprising sodium dodecyl sulfate to a crude polysaccharide solution comprising Neisseria meningitidis selected from serotype A and serotype X, wherein the final concentration of sodium dodecyl sulfate in the solution is 0.1 to less than 1%; b) adding EDTA and sodium acetate to the solution; c) subjecting the solution to alcohol precipitation, followed by centrifugation and retention of supernatant; d) removing the detergent present in the supernatant; e) concentrating and diafiltrating the polysaccharide; wherein the obtained purified polysaccharide from Neisseria meningitidis selected from serotype A and serotype X has a recovery of 60-80%, wherein the removal of detergent is carried out by precipitation of detergent using a potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and combinations thereof.

2. The method of claim 1, wherein a) purified polysaccharide from Neisseria meningitidis serotype A has O-acetylation levels >2 mMol/gm, protein impurity <3%, nucleic acid impurity <1%, and endotoxin impurity <50 EU/μg; and b) purified polysaccharide from Neisseria meningitidis serotype X has protein impurity <1%, nucleic acid impurity <1%, and endotoxin impurity <50 EU/μg.

3. The method of claim 1, wherein the method further comprises the following steps: a) mixing the polysaccharide solution with a cationic detergent; b) subjecting the solution to centrifugation and collection of a pellet; c) dissolving the pellet in alcohol, followed by salt-based precipitation, wherein the cationic detergent is selected from the group consisting of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammoniurn salt, hexadimethrine bromide, and combinations thereof.

4. The method of claim 3, wherein the cationic detergent is cetyltrimethylammonium bromide and the concentration of the cationic detergent in the solution is between 0.1% and 4%.

5. The method of claim 4, wherein the final concentration of cetyltrimethylammonium bromide is in the range of 0.5 to less than 1%.

Description

FIGURES

(1) i. FIG. 1: NMR spectra OF Men-C polysaccharide

(2) ii. FIG. 2: NMR spectra OF Men-Y polysaccharide

(3) iii. FIG. 3: NMR spectra OF Men-W polysaccharide

(4) iv. FIG. 4: NMR spectra OF Men-A polysaccharide

(5) v. FIG. 5: NMR spectra OF Men-X polysaccharide

DESCRIPTION

(6) According to a general aspect of the invention, one of the bacterial serogroup of interest was grown in a suitable medium and inactivated using formaldehyde or any commonly known method in the prior art; further subjecting to downstream processing for isolation of purified capsular polysaccharide. The bacteria of interest for isolation of capsular polysaccharide of the instant invention was obtained from a gram negative bacteria selected from genus including but not limited to Escherichia, Neisseria, Haemophilus, Pseudomonas, etc.; more preferably capsular polysaccharide expressed by serogroup of Neisseria meningitidis. In other aspect of the invention polysaccharide can be derived from group comprising of Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Group A Streptococcus, Group B Streptococcus Ia, Ib, II, III, V, VI, or VIII; Group C Streptococcus, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis, Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis, Salmonella spp., Salmonella typhi, Vibrio cholerae, Pasteurella pestis, Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Hemophilus influenzae, Escherichia coli, Shigella spp., Ehrlichia spp., and Rickettsia spp. Polysaccharides of Streptococcus pneumoniae type 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9A, 9F, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 17F, 18C, 19F, 19A, 20, 22F, 23B, 23F, 24F, 33F, 35B, 38 and 45; Polysaccharides of Neisseria meningitidis serogroup A, B, C, D, W135, X, Y, Z, 29E; and H. influenzae type b.

(7) The biological material used during experimentation were as follows:

(8) Polysaccharides were isolated from:

(9) TABLE-US-00002 TABLE 2 Name of the Strain Organism Designation Source of Strain Neisseria M1027 SynCo Biopartners meningitidis A (Netherlands) Neisseria C11(60E) CBER/FDA, USA meningitidis C Neisseria S877 CBER/FDA, USA meningitidis W Neisseria M10659 CDC, USA meningitidis Y Neisseria M8210 CBER/FDA, USA meningitidis X

(10) Unconjugated carrier protein, i.e. CRM197 or TT. CRM197 was derived from Recombinant Strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA. TT was derived from Clostridium tetani (Harvard No 49205) obtained from CRI, National Control Authority, Kasauli, Himachal Pradesh, India. CRI got this strain from NVI, Netherland.

(11) According to a first embodiment of the invention, bacterial capsular polysaccharide was clarified from inactivated harvest using centrifugation; and the supernatant was subjected to diafiltration using 100 kD tangential flow filtration unit. It is very well understood that any other suitable method may be used in place of centrifugation and diafiltration for the concentration of bacterial capsular polysaccharides by a person skilled in the art. In one of the preferred aspect of this embodiment, capsular polysaccharide was derived from Neisseria meningitidis Serogroup A, C, W, Y & X.

(12) According to a second embodiment of the invention, the retentate obtained in first embodiment was subjected to treatment with anionic surfactant/detergent. The anionic detergent is selected from the group comprising of alkyl sulfates, sodium dodecyl sulfate, 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 .alpha.-olefin sulfonates, sodium alkyl sulfonates, alpha-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and sodium alkyl glyceryl ether sulfonates.

(13) Preferably, said anionic surfactant is an alkyl sulphate, more preferably sodium dodecyl sulphate at a final concentration in the range of 0.1% to 4%, more preferably at 1% was added to the retentate and stirred at room temperature for 2 hour. Amphipathic nature of SDS makes it a strong chaotropic agent that not only disrupts the proteins but also solubilises them.

(14) In another aspect of the second embodiment, inactivated harvest can be directly treated with anionic surfactant and further subjected to concentration of capsular polysaccharide resulting in sufficient reduction of impurities thereby making subsequent step of using cationic detergent dispensable.

(15) According to a third embodiment of the invention, strong alkali was added to the mixture obtained from above embodiment and was adjusted to pH between 9-11 with constant stirring at room temperature for 1 hour. The said strong alkali was selected from a group of comprising of Sodium hydroxide, Potassium Hydroxide, Sodium carbonate, Hydroxyl amine, Triethyl amine and Lithium Hydroxide.

(16) According to a preferred aspect of the third embodiment of the invention, said strong alkali i.e. Sodium hydroxide was added at a final concentration between 5-20M to the mixture obtained from above embodiment and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour.

(17) In another aspect of third embodiment of the invention, alternatively in place of alkali, EDTA and Sodium acetate was added to the mixture obtained from second embodiment, with constant stirring at room temperature for 1 hour.

(18) According to a fourth embodiment of invention, the solution obtained from above embodiment was neutralized (pH 7.0) by addition of a mild organic acid. The said mild organic acid is a combination of one or more acids including formic acid, acetic acid, Propionic acid, Oxalic acid etc. To this neutralized mixture, hydrophilic alcohol, preferably ethanol was added to a final concentration of 30-35% and incubated for couple of hours at room temperature with constant stirring. Hydrophilic alcohol is selected from one of methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone, and t-butyl alcohol; or a combination of two or more thereof; and its concentration is <65% or >95%.

(19) According to a fifth embodiment of invention, excess anionic detergent was removed from the solution. The solution obtained from above embodiment was subjected to centrifugation and supernatant was collected. 0.1M Potassium salt was mixed with the supernatant, and upon its dissolution the mixture was incubated at 2-8° C. for >3 hours. Potassium salt is selected from one of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate, and other potassium salts, or a combination of two or more thereof. This step takes advantage of the low solubility of potassium dodecyl sulphate. Upon addition of potassium salt preferably KCl, the SDS in the solution is converted to potassium dodecyl sulphate, being less soluble and is easily precipitated out, resulting in complete removal of SDS. It is very well understood that the concentration of KCl can be varied, to get the desired result by a person skilled in the art. The protocol as mentioned in this embodiment of the invention may be modified as per requirement by the person skilled in the art.

(20) In another aspect of fifth embodiment, excess anionic detergent can be removed from the solution using Gel filtration, Ethanol precipitation, and Ion exchange resins/Amberlite columns.

(21) According to a sixth embodiment of invention, solution obtained from above embodiment was subjected to centrifugation and supernatant was collected. The supernatant was passed through a 0.2μ filter and retentate was diafiltered through 100 kDa tangential flow filtration.

(22) According to a seventh embodiment of invention, retentate obtained from above embodiment was diafiltered using Tris-HCl buffer at a final concentration of 25 mM. It was followed by addition of a cationic detergent to a final concentration of 1-2% and incubated at RT for 1 hour with constant stirring. The cationic detergent(s) is selected from one of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrine bromide; or a combination of two or more thereof. It is very well understood that the concentration of cationic detergent may be varied in the range of 0.1% to 4%, to get the desired result by a person skilled in the art. Preferably, cationic deteregent is CTAB.

(23) According to an eighth embodiment of invention, solution obtained from above embodiment can be subjected to centrifugation and precipitated CTAB-polysaccharide was collected and dissolved in 30-64% Ethanol. The dissolved mixture can be further subjected to centrifugation for removal of undissolved residues.

(24) According to a ninth embodiment of invention, NaCl was added to a final concentration of 0.1M to the supernatant obtained from above embodiment under constant stirring. The precipitated polysaccharide was collected and dissolved in 1M NaCl followed by alcohol precipitation (30-64%).

(25) According to a tenth embodiment of invention, solution obtained from above embodiment was extensively diafiltered by WFI (Water for injection) using 100 kDa tangential flow filtration and passed through 0.2μ filter and stored at or below −20° C. as final bulk.

(26) According to the eleventh embodiment of the invention the purified capsular polysaccharide of N. meningitidis Serogroup C, W and Y was obtained as per below procedure: Serogroup C, W and Y of N. meningitidis respectively were grown in a suitable medium and inactivated using formaldehyde; Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; Sodium Hydroxide was added to a final concentration of 05-20 mM and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour; Solution obtained from above step was neutralized by addition of mild organic acid i.e. acetic acid. To this neutralized solution, ethanol was added to a final concentration of 30-35% and incubated for couple of hours with constant stirring. Solution obtained from above step was centrifuged and supernatant was collected. 0.1M KCL was added to the supernatant and incubated at 2-8° C. for not less than 3 hour. Solution obtained from above step was subjected to centrifugation and supernatant was collected. The supernatant was passed through a 0.2μ filter and retentate was diafiltered using Tris-HCl buffer through 100 kDa MWCO filter tangential flow filtration. To retentate obtained from the above step, Tris-HCl buffer was added to a final concentration of 25 mM; further followed by addition of CTAB to a final concentration of 2%; and incubated at RT for not less than 1 hour. Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 30-64% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues. To supernatant obtained from above step, NaCl was added to a final concentration of 0.1-0.3 M. The precipitated PS is dissolved in 1M NaCl followed by 30-64% alcohol precipitation. Solution obtained from above step was extensively diafiltered against WFI using 100 KDa tangential flow filtration and passed through 0.2μ filter and stored at −20° C. as final bulk.

(27) According to twelfth embodiment of the invention, purification process as mentioned in eleventh embodiment provides N. meningitidis Serogroup C polysaccharide with a recovery between 60-80%, wherein endotoxin content is less than 50 EU/mg, protein content is less than 0.50% and nucleic acid content less than 0.20%. N. meningitidis Serogroup Y polysaccharide with a recovery between 60-80%, wherein endotoxin content is less than 50 EU/mg, protein content is less than 0.50% and nucleic acid content less than 0.30%. N. meningitidis Serogroup W polysaccharide with a recovery between 60-80%, wherein endotoxin content is less than 50 EU/mg, protein content is less than 0.5% and nucleic acid content less than 0.2%.

(28) According to thirteenth embodiment of the invention the purified capsular polysaccharide of N. meningitidis Serogroup A & X was obtained as per below procedure: Serogroup A & X of N. meningitidis respectively were grown in a suitable medium and inactivated using formaldehyde; Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; EDTA & Sodium acetate were added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; ethanol was added to a final concentration of 30-35% and incubated for couple of hours with constant stirring. Solution obtained from above step was centrifuged and supernatant was collected. 0.1M KCL was added to the supernatant and incubated at 2-8° C. for >3 hours. Solution obtained from above step was subjected to centrifugation and supernatant was collected. The supernatant was passed through a 0.2μ filter and retentate was concentrated & diafiltered using 25 mM Tris-HCl through 100 kDa MWCO tangential flow filtration. CTAB was added to a final concentration of 1-2%; and incubated at RT for not less than 1 hour. Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 30-64% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues. To supernatant obtained from above step, NaCl was added to a final concentration of 0.1-0.3 M. The precipitated PS is dissolved in 1M NaCl followed by 30-64% alcohol precipitation. Solution obtained from above step was extensively diafiltered against WFI using 100 kDa tangential flow filtration and passed through 0.2μ filter and stored at or below −20° C. as final bulk.

(29) According to fourteenth embodiment of the invention, purification process as mentioned in thirteenth embodiment provides N. meningitidis Serogroup A polysaccharide with a recovery between 60-80%, wherein endotoxins content is less than 50 EU/mg, protein content is less than 0.50% and nucleic acid content less than 0.20%. N. meningitidis Serogroup X polysaccharide with a recovery between 60-80%, wherein endotoxins content is less than 50 EU/mg, protein content is less than 0.50% and nucleic acid content less than 0.30%.

(30) According to a fifteenth embodiment of the invention, said purified polysaccharide was conjugated to carrier protein. It is very well understood that the carrier protein used for conjugation with polysaccharides may be any carrier protein known in the art as per requirement by the person skilled in art. The non specific examples of carrier proteins includes carrier protein from a group of but not limited to CRM 197, diphtheria toxoid, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis, detoxified edema factor (EF), 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).

(31) In other aspect of fifteenth embodiment, before conjugation the polysaccharide purified by the instant method was sized by chemical or mechanical means including but not limited to Sonication, microwave, ozonolysis, ionizing radiation, High pressure cell disruption, homogenizer, Microfluidizer, Sodium acetate, sodium metaperiodate, in-vacuo heating etc.

(32) In another aspect of fifteenth embodiment, the polysaccharide purified by the instant method can be conjugated to a carrier protein using reductive amination, cyanylation. Carbodiimide conjugation chemistry.

(33) According to sixteenth embodiment of the invention, immunogenic composition was prepared. The composition comprised of: (a) a conjugate of (i) the capsular saccharide of Neisseria meningitidis serogroup A and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup C and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup Y and (ii) CRM 197; (d) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup W135 and (ii) CRM197; and (e) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup X and (ii) tetanus toxoid.

(34) According to seventeenth embodiment of the invention, endotoxins content can be measured by “Bacterial Endotoxin Test by Kinetic Turbidometric Assay (KTA)” or Rabbit pyrogenicity test; protein content can be measured by Lowry method; and Nucleic acid content can be measured by Spectrophotometry. It is very well understood that any other suitable method may be used for quantification of endotoxin, proteins and nucleic acid content.

(35) In another aspect of seventeenth embodiment, molecular size distribution of purified polysaccharide of Neisseria meningitidis serogroup A, C, W, Y & X can be done using size exclusion high performance liquid chromatography.

(36) In another aspect of sixteenth embodiment, O acetyl content can be measured using colorimetric Hestrin assay.

EXAMPLES

Example 1: Production of Bacterial Polysaccharides (Upstream Process of N. meningitidis Serogroup C)

(37) Using below mentioned fermentation process, polysaccharides were produced.

(38) Fermentation scale: 300 Liters Cleaning in place (CIP), Pressure hold test and Sterilization in place (SIP) of fermenter were performed. After completion of SIP, sterile fermentation medium was aseptically transferred to fermenter. Composition media

(39) Serogroup C Fermentation Media

(40) TABLE-US-00003 TABLE 3 Ingredient Quantity (g/L) D - glucose monohydrate 10 Sodium Chloride 5.8 L-Arqinine 1 L-Serine 1 Calcium Chloride 0.028 Iron(II) Sulfate heptahydrate 0.01 Ammonium Chloride 0.3 Di-potassium Hydrogen Phosphate 4 Yeast Extract 5 Magnesium Chloride 0.4 Soya peptone 3 Casamino Acids 5 Sodium L-glutamate Monohydrate 10 Ammonium Sulphate 1.2 L-Cystine 0.3

(41) Serogroup C Feed Media

(42) TABLE-US-00004 TABLE 4 Ingredient Quantity (g/L) D-glucose monohydrate 200 Sodium L-glutamate Monohydrate 150 Casamino Acids 4 Ammonium chloride 0.2 Dissolved oxygen level was adjusted to desired levels. Fermentation medium was inoculated with culture organism. Fermenter was operated in fed batch mode for 11 to 14 hrs. After the culture reaches desired OD at 590 nm, culture in the fermenter was inactivated using formaldehyde. After completion of incubation, temperature was set to 10±5° C. and followed by cell separation using centrifugation. Supernatant was collected and subjected to depth filtration; clarified harvest was filtered using 0.2μ filter and transferred for purification.
Results:

(43) TABLE-US-00005 TABLE 5 Parameter Men-C Total PS yield of harvest (mg/L) 703.33 Endotoxin Impurity (EU/mg of PS) >500 Protein Impurity (%) 109 Nucleic Acid Impurity (%) 12.3

Example 2: Production of Capsular Polysaccharides (Upstream Process of N. meningitidis Serogroup A, W, X & Y)

(44) Using protocol mentioned in example 1, capsular polysaccharide of N. meningitidis Serogroup A, W, X & Y was produced.

(45) Serogroup Y Fermentation Media

(46) TABLE-US-00006 TABLE 6 Ingredient Quantity (g/L) D - glucose monohydrate 10 Sodium Chloride 5.8 Potassium sulfate 1 L-Arginine 0.75 L-Serine 0.75 L-Cysteine 0.4 Calcium Chloride 0.025 Iron(II) Sulfate heptahydrate 0.01 Ammonium Chloride 0.15 Di-potassium Hydrogen Phosphate 4 Yeast Extract 3 Magnesium Chloride 0.3 Soya peptone 3 Casamino Acid 5 Thiamine hydrochloride 0.05 Sodium L-glutamate Monohydrate 10 L-Tryptophan 0.2
Serogroup Y Feed Media

(47) TABLE-US-00007 TABLE 7 Ingredient Quantity (g/L) D-glucose monohydrate 200 Sodium L-glutamate Monohydrate 150 L-Arginine 1 L-Serine 1 Soya peptone 10 Yeast Extract 16.6
Serogroup W Fermentation Media

(48) TABLE-US-00008 TABLE 8 Ingredient Quantity (g/L) D - glucose monohydrate 10 Sodium Chloride 5.8 Potassium sulfate 1 L-Arginine 0.75 L-Serine 0.75 L-Cysteine 0.4 Calcium Chloride 0.025 Iron(II) Sulfate heptahydrate 0.01 Ammonium Chloride 0.25 Di-potassium Hydrogen Phosphate 4 Yeast Extract 3 Magnesium Chloride 0.3 Soya peptone 3 Casamino Acid 5 Thiamine hydrochloride 0.05 Sodium L-glutamate Monohydrate 10
Serogroup W Feed Media

(49) TABLE-US-00009 TABLE 9 Ingredient Quantity (g/L) D-glucose monohydrate 200 Sodium L-glutamate Monohydrate 150 L-Arqinine 4 L-Serine 4 Soya peptone 4 Yeast Extract 4 Magnesium Chloride 1.6 Calcium Chloride 0.2 Ammonium chloride 0.8
Results:

(50) TABLE-US-00010 TABLE 10 Parameter Men-A Men-X Men-W Men-Y Crude PS (mg/L) 2540 1024 733 1205 Endotoxin (EU/μg OF PS) >500 >500 >500 >500 Protein (%) 19.31 18.2 60.5 38 Nucleic Acid (%) 1.2 1.16 12 3.3

(51) The clarified harvest obtained upon testing, PS (Polysaccharide) content of 1-6 gm/l was observed. This clarified harvest was subjected to further purification.

Example 3: Purification of Capsular Polysaccharides (Downstream Process of N. meningitidis Serogroup C) Using SDS Followed by Alcohol Precipitation

(52) The crude polysaccharide obtained as per example 1 was mixed with varying concentration of SDS. Ethanol was then added to a final concentration which is about 10% below the concentration at which the polysaccharide begins to precipitate. This was further subjected to filtration.

(53) Concentration of SDS tested:

(54) 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%

(55) All the above mentioned concentration of SDS showed efficacy regards to impurity reduction. Above 4% SDS, no significant difference was observed in impurity profile.

(56) Optimal impurity reduction especially proteins was observed at 1% SDS.

(57) The purified polysaccharide had endotoxins impurities >100 EU/μg of polysaccharide, whereas the WHO limit is <100 EU/μg of polysaccharide.

Example 4: Purification of Capsular Polysaccharides (Downstream Process of N. meningitidis Serogroup C)

(58) Protocol:

(59) Two different set of experiments were carried out, 5 Liter and 300 Liter fermentation scale.

(60) Using below mentioned purification process, polysaccharides were purified. Crude polysaccharide obtained as per example 1, was taken in a vessel. Crude polysaccharide was 3-6 fold concentrated by TFF using 100 kDa cassette. Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; Sodium Hydroxide was added to a final concentration of 08-20 mM and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour; Solution obtained from above step was neutralized by addition of Acetic acid. To this neutralized solution, ethanol was added to a final concentration of 33% and incubated for couple of hours with constant stirring. Solution obtained from above step was centrifuged and supernatant was collected. Ethanol concentration was increased to 40%. 0.1M KCL was added to the supernatant and incubated at 2-8° C. for not less than 3 hour. Solution obtained from above step was subjected to centrifugation and supernatant was collected. Polysaccharide was precipitated by increasing final concentration of ethanol to 65%. Polysaccharide pellet was dissolved in 1M NaCl. The supernatant was passed through a 0.2μ filter and retentate was diafiltered using 25 mM Tris-HCl buffer through 100 kDa tangential flow filtration, to obtain purified polysaccharide (Stage I). Addition of CTAB to a final concentration of 2%; and incubated at RT for 1 hour. Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 96% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues. To supernatant obtained from above step, NaCl was added to a final concentration of 0.2M. The precipitated PS is dissolved in 1M NaCl followed by PS precipitation using 65% alcohol. Solution obtained from above step was extensively diafiltered against 0.5M NaCl, followed by WFI using 100 kD tangential flow filtration and passed through 0.2μ filter and stored at −20° C. as final bulk.
Results:

(61) TABLE-US-00011 TABLE 11 Crude purified Purified N. Meningitidis Harvest Polysaccharide Polysaccharide Serogroup C Polysaccharide Stage I Stage II Fermentation 5 L 300 L 5 L 300 L 5 L 300 L Scale PS Volume 1 50 0.65 30 0.6 18 PS 3.87 4.22 3.83 5.62 3.08 7.33 concentration (mg/mL) Total PS 3.87 211 2.48 168 1.84 131.94 (grams) Protein 93.79 109.47 0.6 0.52 0.25 0.13 Impurity (%) Nucleic acid 7.49 12.32 0.02 0.3 0.02 0.08 Impurity (%) Endotoxin NA NA <20 <30 <20 40 Impurity (EU/μg of PS) Step NA NA 64.3 79.9 74.23 78.25 Recovery (%) Molecular NA NA 434 450 392 376 Size by HPLC (kDa) O-Acetyl NA NA 2.8 2.85 2.64 2.6 content Limit: >1.5 mMol/gm Overall NA NA 64.3 79.9 64 62.5 Final Recovery (%) Note: NA = Not applicable

(62) Two different set of experiments were carried out, 5 Litre and 300 Litre fermentation scale.

(63) It was observed that combined use of anionic detergent and alkali had profound effect on reduction of impurity profile. The impurity level at purification stage I & stage II are well below the WHO specification limits for Men-C polysaccharide.

(64) At 5 liters and 300 liters scale, Protein Impurity (%)=<1%; Nucleic acid Impurity (%)=<0.3%; Endotoxin Impurity (EU/μg of PS)=<40EU/μg of PS.

(65) At both scales and at purification stage I & stage II, recovery of purified polysaccharide was above 60% on comparison with crude sample.

Example 5: Purification of Capsular Polysaccharides (Downstream Process of N. meningitidis Serogroup W & Y)

(66) Protocol:

(67) Two different set of experiments were carried out, 5 L and 300 L fermentation scale.

(68) Using below mentioned purification process, polysaccharides were purified. Crude polysaccharide obtained as per example 2, was taken in a vessel. Crude polysaccharide was 3-6 fold concentrated by TFF using 100 kDa cassette. Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; Sodium Hydroxide was added to a final concentration of 08-20 mM and adjusted to pH 10.5 with constant stirring at room temperature for 1 hour; Solution obtained from above step was neutralized by addition of Acetic acid. To this neutralized solution, ethanol was added to a final concentration of 33% and incubated for couple of hours with constant stirring. Solution obtained from above step was centrifuged and supernatant was collected. 0.1M KCL was added to the supernatant and incubated at 2-8° C. for −8 hours. Solution obtained from above step was subjected to centrifugation and supernatant was collected. The supernatant was passed through a 0.2μ filter and retentate was diafiltered using 25 mM Tris-HCl buffer through 100 kDa tangential flow filtration, to obtain purified polysaccharide (Stage I). Addition of CTAB to a final concentration of 2%; and incubated at RT for 1 hour. Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 96% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues. To supernatant obtained from above step, NaCl was added to a final concentration of 0.25M. The precipitated PS is dissolved in 1M NaCl followed by PS precipitation using 65% alcohol. Solution obtained from above step was extensively diafiltered against WFI using 100 kDa tangential flow filtration and passed through 0.2μ filter and stored at −20° C. as final bulk (Stage II).
Result:

(69) TABLE-US-00012 TABLE 12 Crude purified Purified N. Meningitidis Harvest Polysaccharide Polysaccharide Serogroup Y Polysaccharide Stage I Stage II Fermentation 5 L 300 L 5 L 300 L 5 L 300 L Scale PS Volume 1.25 50 0.75 35 0.8 29.5 PS 5.67 7.23 7.23 7.88 5.39 8.41 concentration (mg/mL) Total PS 7.08 361.5 5.42 275.8 4.31 248 (grams) Protein 38.62 38.03 2.21 1.64 0.16 0.08 Impurity (%) Nucleic acid 1.94 3.31 0.69 0.25 0.11 0.71 Impurity (%) Endotoxin NA NA 50 56 44 45 Impurity (EU/μg of PS) Step NA NA 76.50 76.29 79.52 89.95 Recovery (%) Molecular NA NA 660 620 608 543 Size by HPLC (kDa) O-Acetyl NA NA 0.85 0.8 0.79 0.75 content Limit: >0.3 mMol/gm Overall NA NA 76.50 76.29 60.83 68.62 Final Recovery (%)

(70) TABLE-US-00013 TABLE 13 Crude purified Purified N. Meningitidis Harvest Polysaccharide Polysaccharide Serogroup W Polysaccharide Stage I Stage II Fermentation 5 L 300 L 5 L 300 L 5 L 300 L Scale PS Volume 1 39 0.8 28.5 0.8 16.5 PS 4.26 5.65 3.5 6.01 3.25 8.75 concentration (mg/mL) Total PS 4.26 220 2.8 171 2.6 144 (Grams) Protein 62.1 60.53 3.4 2.16 <0.21 <0.21 Impurity (%) Nucleic acid 7.98 12 0.85 1.16 0.1 0.09 Impurity (%) Endotoxin NA NA <30 <40 <20 <10 Impurity (EU/μg of PS) Step NA NA 65 77 92 84.2 Recovery (%) Molecular NA NA 400 420 395 380 Size by HPLC (kDa) O-Acetyl NA NA 1.21 1.15 1.05 0.99 content Limit: >0.3 mMol/gm Overall NA NA 65 77 61 65.4 Final Recovery (%)

(71) Two different set of experiments were carried out, 5 L and 300 L fermentation scale.

(72) On comparison, it was observed that the impurity level at purification stage I & stage II are well below the WHO specification limits for Men-Y & Men-W polysaccharide.

(73) It was observed that combined use of anionic detergent and alkali had profound effect on reduction of impurity profile. The impurity level at purification stage I & stage II are well below the WHO specification limits for Men-C polysaccharide.

(74) At 5 L and 300 L scale, Protein Impurity (%)=<3.5%; Nucleic acid Impurity (%).sub.7<1.5%; Endotoxin Impurity (EU/μg of PS)=<60EU/μg of PS.

(75) At both scales and at purification stage I & stage II, recovery of purified polysaccharide was above 60% on comparison with crude sample.

Example 6: Purification of Capsular Polysaccharides (Downstream Process of N. meningitidis Serogroup A & X)

(76) Protocol:

(77) Two different set of experiments were carried out, 5 L and 300 L fermentation scale.

(78) Using below mentioned purification process, polysaccharides were purified. Crude polysaccharide obtained as per example 2, was taken in a vessel. Crude polysaccharide was 3-6 fold concentrated by TFF using 100 kDa cassettes. Sodium Dodecyl Sulphate was added to this inactivated harvest to a final concentration of 1% and stirred at room temperature for 2 hour; Sodium Acetate, EDTA & Sodium Dodecyl Sulphate was added to a final concentration of 6%, 2 mM & 1% respectively; with constant stirring at room temperature for 1 hour; Solution obtained from above step was centrifuged and supernatant was collected. 0.1M KCL was added to the supernatant and incubated at 2-8° C. for −3 hours. Solution obtained from above step was subjected to centrifugation and supernatant was collected. The supernatant was passed through a 0.2μ filter and retentate was diafiltered using 25 mM Tris-HCl buffer through 100 kDa tangential flow filtration, to obtain purified polysaccharide (stage I). Addition of CTAB to a final concentration of 2%; and incubated at RT for 1 hour. Solution obtained from above step was centrifuged and precipitate was collected and dissolved in 96% Ethanol. The dissolved mixture was further subjected to centrifugation for removal of undissolved residues. To supernatant obtained from above step, NaCl was added to a final concentration of 0.2M. The precipitated PS is dissolved in 1M NaCl followed by 65% alcohol precipitation. Solution obtained from above step was extensively diafiltered against WFI using 100 kD tangential flow filtration and passed through 0.2μ filter and stored at −20° C. as final bulk (stage II).
Results:

(79) TABLE-US-00014 TABLE 14 Crude purified Purified N. Meningitidis Harvest Polysaccharide Polysaccharide Serogroup A PS Stage I Stage XI Fermentation 300 L 300 L 300 L Scale PS Volume 50 45 30 PS concentration 11.44 6.63 9.75 (mg/mL) Total PS (grams) 572 298.35 292.5 Protein 19.31 2.05 0.14 Impurity (%) Nucleic acid 1.22 0.12 0.05 Impurity (%) Endotoxin Impurity NA 29.08 35 (EU/μg of PS) Residual SDS (ppm) NA <5 <5 Step Recovery (%) NA 52 98 Molecular Size NA 350 306 by HPLC (kDa) O-Acetyl content NA 2.69 2.65 Limit: >2 mMol/gm Overall Final NA 52 51 Recovery (%)

(80) TABLE-US-00015 TABLE 15 Crude purified Purified N. Meningitidis Harvest Polysaccharide Polysaccharide Serogroup X PS Stage I Stage II Fermentation 300 L 300 L 300 L Scale PS Volume 55 40 30 PS concentration 5.05 5.23 6.21 (mg/mL) Total PS (grams) 277.75 209.2 186.30 Protein 18.21 0.6 0.54 Impurity (%) Nucleic acid 1.16 0.76 0.14 Impurity (%) Endotoxin Impurity NA 30 13.79 (EU/μg of PS) Residual SDS (ppm) NA <5 <5 Step Recovery (%) NA 75.3 88.9 Molecular Size NA 485 479 by HPLC (kDa) Overall Final NA 75.3 67.25 Recovery (%)

(81) On comparison, it was observed that the impurity level at purification stage I & stage II are well below the WHO specification limits for Men-A & Men-X polysaccharide.

(82) It was observed that use of anionic detergent had profound effect on reduction of impurity profile. The impurity level at purification stage I & stage II are well below the WHO specification limits for Men-A & Men-X polysaccharide.

(83) At 300 L scale, Protein Impurity (%)=<2.5%; Nucleic acid Impurity (%)=<1%; Endotoxin Impurity (EU/μg of PS)=<40 EU/μg of PS.

(84) At both scales and at purification stage I & stage II, recovery of purified polysaccharide of serogroup X was above 60% and purified polysaccharide of serogroup A was above 50%, on comparison with crude sample.

Example 7

(85) Structural Integrity of the Isolated Polysaccharides (PS)

(86) Refer FIGS. 1-5 for NMR spectra.

(87) Structural integrity of the isolated polysaccharides of N. meningitidis serogroup A, C, W, Y & X was verified using Nuclear Magnetic Resonance (NMR.

(88) NMR spectra recorded were comparable and confirmed their identity as partially O-acetylated Meningococcal polysaccharide of serogroup A, C, W, & Y; and N-acetylated polysaccharide of serogroup X.

Example 8

(89) Comparative analysis of Sodium Deoxycholate (DOC) based purification process and the claimed process for purification of polysaccharides.

(90) Sodium Deoxycholate (DOC) based process “An improved method for meningococcus polysaccharide purification Tanizaki et al (Conjugate and Polysaccharide Vaccines, Poster 79, http://neisseria.org/ipnc/1996/Neis1996-chap4.pdf)” was optimized as follows:

(91) 100 kD Harvest; DOC+EDTA+NaOAc+ethanol (40%) treatment; Centrifugation, collection of supernatant and 0.2μ filtration; Concentration and Diafilration; CTAB (3%) treatment at RT; Centrifugation and collection of CTAB-PS pellet; Dissolution of CTAB-PS pellet in 96% ethanol and precipitation by 0.1 M NaCl; Dissolution of precipitate in 40% ethanol and addition of 1 M NaCl (2-8° C.); Centrifugation and collection of supernatant; Carbon filtration; Precipitation of PS by increasing ethanol concentration; PS pellet dissolution in WFI and concentration and diafilration with WFI and storage at −20° C. after 0.2μ filtration.

(92) The DOC based process is being widely used across industry for purification of polysaccharides to be used as vaccine antigen.

(93) The impurity profile of polysaccharide obtained by DOC based method was compared with the claimed inventive process.

(94) Results:

(95) TABLE-US-00016 TABLE 16 % % Final % Nucleic Endotoxin Recovery protein acid (EU/μg of PS) Men-A DOC 65 <0.3 <0.2 <20 SDS 64 <0.3 <0.2 <20 Men-C DOC 65 <0.3 <0.2 <20 SDS 62.00 <0.3 <0.2 <20 Men-W DOC 60 <0.5 <0.5 <30 SDS 70 <0.5 <0.5 <30 Men-Y DOC 60 <0.5 <0.5 <30 SDS 70 <0.5 <0.5 <30 Men-X DOC 65 <0.3 <0.2 <20 SDS 70 <0.3 <0.2 <20

(96) SDS based inventive method and the DOC based method showed similar results for serogroup A & C, whereas for serogroup W, Y & X SDS method showed improved final recovery as compared to DOC method.

(97) As can be seen by the above results the modified SDS process has clear advantages in terms of both, ease of operation as well as several other advantage such as, DOC being an animal origin component and is non HALAL compliant. DOC is manufactured & supplied by a single vendor throughout the world, whereas SDS is a synthetic detergent, HALAL certified and with several suppliers available globally. DOC breaks down the endotoxins without effecting the chemical composition and once the detergent is removed it can regain its biological activity, whereas SDS owing to its amphipathic nature and higher aggregation number denatures and solubilizes the proteins well and also disrupts endotoxins irreversibly to its monomeric units.

(98) By using SDS a decreased consumption of chemicals and consumables (ethanol, ultra-filters, carbon filters etc) has a distinct advantage. Additionally, use of SDS as a replacement was non-invasive on structural integrity of the polysaccharides.

Example 9

(99) Immunogenic composition as shown below was prepared and tested for immunogenicity.

(100) The composition comprised of: (a) a conjugate of (i) the capsular saccharide of Neisseria meningitidis serogroup A and (ii) tetanus toxoid; (b) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup C and (ii) CRM197; (c) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup Y and (ii) CRM 197; (d) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup W135 and (ii) CRM197; and (e) a conjugate of (i) capsular saccharide of Neisseria meningitidis serogroup X and (ii) tetanus toxoid.
Results:

(101) The immunogenic composition was found to be immunogenic.

Advantages of the Invention

(102) The process results in significant reduction of endotoxin, protein and nucleic acid impurities thereby providing higher recovery of capsular polysaccharide with the desired O-acetyl levels. As can be seen by the above results the claimed process has clear advantages in terms of ease of operation, as well as several other advantage such as, Regulatory issue: DOC is an animal origin component and is non HALAL compliant. It is manufactured & supplied by a single vendor throughout the world, whereas SDS is a synthetic detergent, HALAL certified and with several suppliers available globally. Functional issue: DOC breaks down the endotoxins without effecting the chemical composition and once the detergent is removed it can regains its biological activity, whereas SDS owing to its amphipathic nature and higher aggregation number denatures and solubilizes the proteins well and also disrupts endotoxins irreversibly to its monomeric units By using SDS a decreased consumption of chemicals and consumables (ethanol, ultra-filters, carbon filters etc) was a distinct advantage. Inventive process of the present patent application results in significant decrease of other cellular impurities. Further, the process provides effective removal of reagents used during the upstream and downstream processing. Additionally, Inventive process of the present patent application was non-invasive on structural integrity of the polysaccharides, thereby maintaining its immunogenicity.

(103) In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.