WE CLAIM:
1. A method for quantification of O-specific polysaccharide content in a sample, the
method comprising:
a) adding a chromogen to the sample to obtain a mixture, wherein the sample is
5 optionally diluted with a buffer prior to the addition of the chromogen;
b) mixing an acid to the mixture to obtain a solution;
c) incubating the solution;
d) subjecting the incubated solution to a spectrophotometric assay or a colorimetric
assay; and
10 e) quantifying the O-specific polysaccharide content in the sample using a mixed
saccharide standard solution,
wherein the mixed saccharide standard solution comprises at least two
saccharides.
15 2. The method as claimed in claim 1, wherein the buffer is selected from a group
comprising tris, histidine, pyridine, citrate, phosphate, acetate, disodium phosphate,
monopotassium phosphate, sodium chloride, sodium borate, succinic acid, nitrate,
sodium hydroxide, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), PIPES
(piperazine-N,N′-bis(2-ethanesulfonic acid), CHAPS (3-[(3-
20 cholamidopropyl)dimethylammonio]-1-propanesulfonate), MOPS (3-(Nmorpholino)propanesulfonic acid), Bicine, bis-Tris, carbonate, Tricine, ACES (N-(2-
acetamido)-2-aminoethanesulfonic acid), or any combination thereof.
3. The method as claimed in any one of claims 1 or 2, wherein the chromogen is selected
25 from a group comprising phenol, resorcinol, 2,6-dimethylphenol, orcinol, DNSA (3,5-
dinitrosalicylaldehyde), TNBS (2,4,6-Trinitrobenzene sulfonic acid), or any combination
thereof, at a concentration ranging from about 70% to 90%.
4. The method as claimed in any one of claims 1 to 3, wherein the acid is selected from a
30 group comprising hydrochloric acid, sulphuric acid, perchloric acid, carbonic acid
hydrofluoric acid, phosphoric acid, trifluoroacetic acid, acetic acid, nitric acid, or any
combination thereof, at a concentration ranging from about 15 M to 20 M.
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5. The method as claimed in any one of claims 1 to 4, wherein the solution is incubated in
step c) at a temperature ranging from about 20 °C to 30 °C, for a duration ranging from
about 20 minutes to 50 minutes.
5 6. The method as claimed in any one of claims 1 to 5, wherein the mixed saccharide
standard solution comprises at least two saccharides selected from a group comprising
monosaccharide, disaccharide, oligosaccharide, polysaccharide, or any combination
thereof;
optionally, wherein the mixed saccharide standard solution comprises at least two
10 monosaccharides selected from a group comprising glyceraldehyde (triose), ribose
(pentose) glucose (hexose), fructose (hexose), galactose (hexose), tagatose, mannose,
arabinose, xylose, erythrose (tetrose), sedoheptulose (heptose), rhamnose, paratose, or
any combination thereof.
15 7. The method as claimed in claim 6, the mixed saccharide standard solution comprises
rhamnose, mannose, galactose, glucose and paratose in a ratio of about 1: 1: 1: 0.76:1.
8. The method as claimed in claim 1, wherein the sample is selected from a group
comprising a purified O-specific polysaccharide, a purified O-specific polysaccharide
20 protein conjugate bulk, or a vaccine formulation including O-specific polysaccharide
protein conjugate.
9. The method as claimed in claim 8, wherein the sample comprising O-specific
polysaccharide protein conjugate is subjected to pre-treatment prior to the dilution of the
25 sample, for removal of excipients selected from a group comprising maltose, lactose,
sucrose, mannitol and trehalose;
optionally, wherein the pre-treatment comprises passing the sample to desalting
column, membrane filter, or any combination thereof.
30 10. The method as claimed in claim 9, wherein the desalting column is selected from a group
comprising cross linked dextran based, polysaccharide based, porous polyacrylamide
based and polypropylene based.
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11. The method as claimed in claim 9, wherein the membrane filter is selected from a group
comprising polyethersulfone based, modified polyethersulfone based, regenerated
cellulose based, and water-wettable polytetrafluoroethylene based; wherein the
membrane has a molecular weight cut-off (MWCO) from about 1 to 10 kDa.
5
12. The method as claimed in any one of claims 8 to 11, wherein the sample comprising
purified O-specific polysaccharide protein conjugate bulk and vaccine formulation is
treated with a protein precipitating agent, to separate free or unconjugated O-specific
polysaccharide and conjugated O-specific polysaccharide;
10 wherein the protein precipitating agent is selected from a group comprising
trichloroacetic acid, deoxycholate-hydrochloric acid (DOC-HCl), ethanol, acetone,
ammonium sulfate, PEG8000, diethyl ether, or any combinations thereof.
13. The method as claimed in any one of claims 1 to 12, wherein the O-specific
15 polysaccharide content in the sample is free O-specific polysaccharide content and total
O-specific polysaccharide content.
14. The method as claimed in any one of claims 1 to 13, wherein the O-specific
polysaccharide is selected from a group of Gram-negative bacteria comprising:
20 Salmonella spp., Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis,
Salmonella typhimurium; Shigella spp., Shigella sonnei, Shigella flexneri, Shigella
dysenteriae, Citrobacter spp., C. freundii, C. werkmanii; Cronobacter species (former
Enterobacter sakazakii); Shigella boydii; Escherichia coli; Klebsiella pneumoniae;
Vibrio cholerae; Pseudomonas aeruginosa; Plesiomonas shigelloides; or Campylobacter
25 jejuni.
15. A method for quantification of O-specific polysaccharide content in a sample, said
method comprising:
i. diluting the sample with a buffer to obtain the diluted sample comprising the O30 specific polysaccharide in the range from about 1 µg/ml to 50.0 µg/ml;
ii. adding phenol in the range from about 70.0% to 90.0% to the diluted sample to
obtain a mixture;
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iii. mixing sulphuric acid in range from about 15.0 M to 20.0 M to the mixture to
obtain a solution;
iv. incubating the solution at temperature in range from about 20.0 °C to 30.0 °C for
duration in range of about 20.0 minutes to 50.0 minutes;
5 v. subjecting the incubated solution to a spectrophotometric assay at 480 nm;
vi. quantifying the O-specific polysaccharide content in the sample using a mixed
saccharide standard solution;
wherein the mixed saccharide standard solution comprises at least two
saccharides; and
10 wherein the sample is a purified O-specific polysaccharide, a purified Ospecific polysaccharide protein conjugate bulk, or a vaccine formulation; and
wherein the purified O-specific polysaccharide protein conjugate bulk and
final vaccine formulation is additionally subjected to pre-treatment by passing
through a desalting column and a membrane filter and treating with DOC-HCl,
15 prior to the dilution of the sample.