WO2011148382A1 - An improved process for the purification of capsular polysaccharides of haemophilus influenza - b, neisseria meningitis such as serotypes a, c, y and w-135, and other similar related capsular polysaccharides produced from both gram negative and gram positive microorganisms using aluminium phosphate with alcohol. - Google Patents
An improved process for the purification of capsular polysaccharides of haemophilus influenza - b, neisseria meningitis such as serotypes a, c, y and w-135, and other similar related capsular polysaccharides produced from both gram negative and gram positive microorganisms using aluminium phosphate with alcohol. Download PDFInfo
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- WO2011148382A1 WO2011148382A1 PCT/IN2010/000476 IN2010000476W WO2011148382A1 WO 2011148382 A1 WO2011148382 A1 WO 2011148382A1 IN 2010000476 W IN2010000476 W IN 2010000476W WO 2011148382 A1 WO2011148382 A1 WO 2011148382A1
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- Prior art keywords
- capsular polysaccharides
- polysaccharide
- serotypes
- pellet
- added
- Prior art date
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 63
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 63
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 44
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 238000000746 purification Methods 0.000 title claims abstract description 16
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 title claims abstract description 15
- 244000005700 microbiome Species 0.000 title claims abstract description 12
- 241000606768 Haemophilus influenzae Species 0.000 title claims abstract description 10
- 241000588653 Neisseria Species 0.000 title claims description 14
- 201000009906 Meningitis Diseases 0.000 title claims description 7
- 229940001007 aluminium phosphate Drugs 0.000 title description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 title description 2
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 108010060123 Conjugate Vaccines Proteins 0.000 claims abstract description 3
- 229940031670 conjugate vaccine Drugs 0.000 claims abstract description 3
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract 2
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 19
- 239000008188 pellet Substances 0.000 claims description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 13
- 238000011534 incubation Methods 0.000 claims description 13
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- 239000002158 endotoxin Substances 0.000 claims description 8
- 239000012535 impurity Substances 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 229960005486 vaccine Drugs 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 230000021615 conjugation Effects 0.000 claims description 4
- 239000010414 supernatant solution Substances 0.000 claims description 4
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000000356 contaminant Substances 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 229960003964 deoxycholic acid Drugs 0.000 claims 8
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims 6
- BDDLHHRCDSJVKV-UHFFFAOYSA-N 7028-40-2 Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O BDDLHHRCDSJVKV-UHFFFAOYSA-N 0.000 claims 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims 1
- 230000001376 precipitating effect Effects 0.000 claims 1
- 239000011541 reaction mixture Substances 0.000 claims 1
- 229940031937 polysaccharide vaccine Drugs 0.000 abstract description 3
- 241000588650 Neisseria meningitidis Species 0.000 abstract 1
- 229940124856 vaccine component Drugs 0.000 abstract 1
- 206010021198 ichthyosis Diseases 0.000 description 14
- 239000006228 supernatant Substances 0.000 description 8
- 241000606790 Haemophilus Species 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000001556 precipitation Methods 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 238000011026 diafiltration Methods 0.000 description 3
- 238000012869 ethanol precipitation Methods 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100028085 Glycylpeptide N-tetradecanoyltransferase 1 Human genes 0.000 description 2
- 101710081880 Glycylpeptide N-tetradecanoyltransferase 1 Proteins 0.000 description 2
- 241000709721 Hepatovirus A Species 0.000 description 2
- 241000709701 Human poliovirus 1 Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 208000005252 hepatitis A Diseases 0.000 description 2
- 229960001438 immunostimulant agent Drugs 0.000 description 2
- 239000003022 immunostimulating agent Substances 0.000 description 2
- 230000003308 immunostimulating effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 206010027202 Meningitis bacterial Diseases 0.000 description 1
- 241000921898 Neisseria meningitidis serogroup A Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 201000009904 bacterial meningitis Diseases 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 229940001442 combination vaccine Drugs 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960003983 diphtheria toxoid Drugs 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011369 resultant mixture Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 238000009283 thermal hydrolysis Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates an improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, such as serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, more particularly in the field of new vaccines development, immunology, and medicine and for the prevention of infection by bacterial pathogens having protective polysaccharide capsule, by immunization
- US Patent 7588765 discloses about the polysaccharide and glycoconjugate vaccines, more particularly the published prior art relates to a conjugation process which involves activation of an endotoxin-free polysaccharide antigen to polyfunctional capsular polysaccharide through a diamino-alkyl spacer introduced.
- a conjugation process which involves activation of an endotoxin-free polysaccharide antigen to polyfunctional capsular polysaccharide through a diamino-alkyl spacer introduced.
- an aluminum hydroxide and aluminum phosphate had been used proportionately adjusting conjugation pH
- Patent application number: 20100040647 which teaches about the method of making a vaccine (i.e. a combination vaccine) using inactivated poliovirus type 1 , comprising the steps of: mixing diphtheria toxoid and tetanus toxoid, followed by the addition of inactivated poliovirus type 1 at a dose greater than 10 D-antigen units and less than 20 D-antigen units and optionally IPV type(s) 2 and/or 3 (IPV).
- a vaccine i.e. a combination vaccine
- IPV type(s) 2 and/or 3 IPV
- Patent 7357936 which teaches about the process for the manufacture of a vaccine composition comprising admixing a) an adjuvant composition containing an immunostimulant adsorbed onto a first metallic salt particle substantially free of antigen, and b) an antigen, wherein the antigen is adsorbed onto a second metallic salt particle substantially free of immunostimulant, wherein the metallic salt of each of the first metallic salt particle and the second metallic salt particle may be the same.
- EP00024 disclose the method which comprises the use of toxic components like Phenol, organic solvents like isopropanol, ethanol, use of detergents like CTAB for polysaccharide precipitation and the use of sodium acetate for pH adjustments,
- EPO 497525 discloses the method which involves thermal hydrolysis and sodium acetate to hydrolyze the sample.
- CA 1206905 describes a method which uses toxic and organic solvents like Phenol, butanol, Toluene and chloroform and use of detergent Cetavlon (CTAB).
- US 20060228380 discloses a method which comprises Cetavlon for polysaccharide precipitation, carbon filter for nucleic acid removal and potassium iodide for precipitation of Cetavlon [10, 1 1], US Patent 5,847,1 12 mentions a method which makes use of multiple isopropyl alcohol precipitations and Cetavlon for the precipitation of PS.
- Important object of the present invention is that an improved process is a simple, efficient and improved purification process that eliminates the impurities from the above mentioned polysaccharides meeting the specifications as per the compendial requirements.
- the significant finding of this invention is applicable to Haemophilus influenza- b and Neisseria such as menigitides serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms which are used in polysaccharide conjugate vaccine manufacturing.
- Further important object of the present invention is that a process can purify polysaccharides robustly at commercial scale, requiring low cost materials in the process and no specialized facilities or disposal of hazardous materials, therefore an alleged invention is cost effective. Further very important object of the present invention is that the present process is very effective in preparing a high quality product with high yields. And very much effective to remove impurities by use of aluminium phosphate from the capsular polysaccharides meeting the compendial requirements.
- A1P04 in combination of alcohol and DOC buffer is used for the removal of impurities.
- the process is a generic process to purify Haemophilus influenzae -b, Neisseria serotypes A, C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms.
- the present invention relates to an improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis such as serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, and the use of alcohol / DOC during Haemophilus and Neisseria polysaccharides purification and the inefficiency in the prior art process in removal of endotoxin, the present invention makes use of aluminum phosphate and DOC buffer for removal of endotoxin, alcohol / DOC treatment for nucleic acid and protein removal very effectively.
- Impurities in all Haemophilus and Neisseria polysaccharide types are effectively adsorbed with aluminum phosphate in presence of alcohol without significant loss to the polysaccharide content.
- An alleged invention is an alcohol based purification process along with aluminum phosphate and DOC buffer for Haemophilus and Neisseria polysaccharides which meet all the release test specifications required for the vaccination of adults Haemophilus and Neisseria type cultures, used in this invention are available worldwide from a great number of culture libraries.
- the present invention relates to a process for preparing a purified Haemophilus polysaccharide the said process comprises the following steps ras shown in flow chart - 1.
- the primary step of the present invention for purification of polysaccharide is Cetavlon precipitation
- the above mixture is centrifuged at 6000 RPM at 2-8°C for 30 minutes to recover supernatant.
- Added sodium acetate to the supernatant to a final concentration of 6% w/v & pH is adjusted to 6.3 ⁇ O. l using 8M Acetic acid, then added ethanol to the solution to a final concentration of 64% v/v under agitation adjusted the pH to 6.8 ⁇ 0.1 using 8M Acetic Acid, with or without incubation .
- incubation is preferred, the solution is precipitated at 2°C - 8°C for 2-4hrs.
- the product is characterized using sup. l H-NMR data shows that the consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule.
- The.1 H-NMR spectrum showed a series of well-resolved signals (protons from the methyl group) for the quantification of functional groups in the polysaccharide.
- Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide.
- the purified polysaccharides comply with the WHO specifications for pure polysaccharides. WHO Specifications for Purified polysaccharides:
- Protein Content - NMT 1% dry weight of the polysaccharide.
- Nucleic acid Content - NMT 1% dry weight of the polysaccharide.
Abstract
A manufacturing process for preparing pure capsular polysacharide using aluminum phosphate with alcohol for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitidis such as serotypes A, C, Y, W- 135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms is described. The process is applicable, simple, robust and easily scalable. The purified polysaccharide can be used in the preparation of covalent conjugates comprising the polysaccharide linked to a carrier protein. Purified polysaccharides meet all the specifications required by WHO and can be used as a vaccine component for both polysaccharide and conjugate vaccines.
Description
fiield of the invention
The present invention relates an improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, such as serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, more particularly in the field of new vaccines development, immunology, and medicine and for the prevention of infection by bacterial pathogens having protective polysaccharide capsule, by immunization
Back Ground of the invention:-
US Patent 7588765 discloses about the polysaccharide and glycoconjugate vaccines, more particularly the published prior art relates to a conjugation process which involves activation of an endotoxin-free polysaccharide antigen to polyfunctional capsular polysaccharide through a diamino-alkyl spacer introduced. In the said conjugation process an aluminum hydroxide and aluminum phosphate had been used proportionately adjusting conjugation pH
Patent application number: 20100040647, which teaches about the method of making a vaccine (i.e. a combination vaccine) using inactivated poliovirus type 1 , comprising the steps of: mixing diphtheria toxoid and tetanus toxoid, followed by the addition of inactivated poliovirus type 1 at a dose greater than 10 D-antigen units and less than 20 D-antigen units and optionally IPV type(s) 2 and/or 3 (IPV).
US Patent 699192 The said published prior art discloses about a process for the production of inactivated Hepatitis A virus substantially free of host cell contamination, comprising the following steps of :
a) a culturing Hepatitis A virus and harvesting a hepatitis A preparation;
b) treating said hepatitis A preparation with a protease, thereafter;
c) separating intact virus from protease-digested protein; and
d) inactivating said virus
S Patent 7357936 , which teaches about the process for the manufacture of a vaccine composition comprising admixing a) an adjuvant composition containing an immunostimulant adsorbed onto a first metallic salt particle substantially free of antigen, and b) an antigen, wherein the antigen is adsorbed onto a second metallic salt particle substantially free of immunostimulant, wherein the metallic salt of each of the first metallic salt particle and the second metallic salt particle may be the same.
The following problems are associated with the prior art, that the polysaccharide vaccines have been licensed for many years and have proved valuable in preventing bacterial meningitis in high risk individuals. Despite of several studies carried out on these encapsulated bacteria, there is a lack of information in the literature related to large- scale processes of bacterial cultivation and removal of impurities from capsular polysaccharides [3, 4]·
EP00024 disclose the method which comprises the use of toxic components like Phenol, organic solvents like isopropanol, ethanol, use of detergents like CTAB for polysaccharide precipitation and the use of sodium acetate for pH adjustments,
Furthermore, EPO 497525 discloses the method which involves thermal hydrolysis and sodium acetate to hydrolyze the sample.
CA 1206905 describes a method which uses toxic and organic solvents like Phenol, butanol, Toluene and chloroform and use of detergent Cetavlon (CTAB).
US 4242501 and some more studies mention a method which involves serotype specific anion exchange chromatography [5]. Classical methods described by Institute Merieux give details of the use of Cetavlon and protease [6] for purification of N. Meningitidis serogroup A, C polysaccharide purification [7]. Some other methods made use of proteinase for removal of proteins and use of lectin agarose columns resulting in high costs and process which can't be scaled up easily [8, 9].
US 20060228380 discloses a method which comprises Cetavlon for polysaccharide precipitation, carbon filter for nucleic acid removal and potassium iodide for precipitation of Cetavlon [10, 1 1],
US Patent 5,847,1 12 mentions a method which makes use of multiple isopropyl alcohol precipitations and Cetavlon for the precipitation of PS.
The above disclosed prior arts teach about a method which is not efficient in removing the endotoxin from capsular polysaccharides of bacteria if the initial load is high and if produced in aggregate forms. The published prior art do not teaches about the use of aluminum phosphate in combination with DOC, EDTA etc, which is an efficient process to purify the capsular polysaccharides and complete removal of endotoxin during the process.
An inventive feature that the use of aluminum phosphate along with alcohol has not been reported in any kind of published documents, and a use of Aluminum phosphate along with alcohol achieved upto the mark of purification of capsular polysaccharide which is also reduces cost of manufacturing as well. An improvement in a process using a new ingredient is an inventive over the prior art an improved process is not obvious in the filed of purification of polysaccharide technology, therefore the present invention is novel and inventive over the prior art.
Objects of the invention :-
Important object of the present invention is that an improved process is a simple, efficient and improved purification process that eliminates the impurities from the above mentioned polysaccharides meeting the specifications as per the compendial requirements. The significant finding of this invention is applicable to Haemophilus influenza- b and Neisseria such as menigitides serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms which are used in polysaccharide conjugate vaccine manufacturing.
Further important object of the present invention is that a process can purify polysaccharides robustly at commercial scale, requiring low cost materials in the process and no specialized facilities or disposal of hazardous materials, therefore an alleged invention is cost effective.
Further very important object of the present invention is that the present process is very effective in preparing a high quality product with high yields. And very much effective to remove impurities by use of aluminium phosphate from the capsular polysaccharides meeting the compendial requirements.
Summary of the invention
An improved process for purification of polysachharide using an alcohol / A1P04 method for the purification of Haemophilus and Neisseria such as serotypes A, C, Y and W - 135, other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms which are used in the preparation of polysaccharide vaccines. A1P04 in combination of alcohol and DOC buffer is used for the removal of impurities. The process is a generic process to purify Haemophilus influenzae -b, Neisseria serotypes A, C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms.
Description of the Invention
The present invention relates to an improved process for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis such as serotypes A,C, Y and W-135, and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, and the use of alcohol / DOC during Haemophilus and Neisseria polysaccharides purification and the inefficiency in the prior art process in removal of endotoxin, the present invention makes use of aluminum phosphate and DOC buffer for removal of endotoxin, alcohol / DOC treatment for nucleic acid and protein removal very effectively. Impurities in all Haemophilus and Neisseria polysaccharide types are effectively adsorbed with aluminum phosphate in presence of alcohol without significant loss to the polysaccharide content. An alcohol / DOC treatment efficient for removal of all the nucleic acids and protein contaminants. An alleged invention is an alcohol based purification process along with aluminum phosphate and DOC buffer for Haemophilus and Neisseria polysaccharides which meet all the release test specifications required for the vaccination of adults Haemophilus and Neisseria type cultures, used in this invention are available worldwide from a great number of culture libraries.
The American Type Culture Collection (ATCC), 12301 Parklawn Dr., Rockville, Md., U.S.A.
20852, lists all of the serotypes of this invention as being freely available.
Therefore, the present invention relates to a process for preparing a purified Haemophilus polysaccharide the said process comprises the following steps ras shown in flow chart - 1.
Flow chart 1:
The primary step of the present invention for purification of polysaccharide is Cetavlon precipitation
Taken the crude PRP in the cold room and centrifuge at 6000 RPM / 30 min / 2 - 8 °C, after centrifugation discarded the pellets formed, collected the supernatant solution and added 10% Cetavlon to a concentration of 0.65 %, the resultant mixture is allowed to incubate at room temperature under constant stirring for a period of two hours.
0.5 M NaCl dissolution:
After completion of incubation time the above mixture is allowed to centrifuge at 4000 RPM at 1 to 10c for 15 to 35 minutes to recover pellet. The said pellet is dissolved in 0.5 M NaCl (Make up the volume to initial crude volume). This solution is filtered through 0.45μ filter.
72% Ethanol precipitation:
Added 72% Ethanol to the above filtrate to get a final concentration of 72%. The addition of ethanol is carried out under constant stirring. Adjusted the pH to 6.8 ± 0.1 using 8M acetic acid, stored the precipitated solution at room temperature for 2 - 4 hrs.
DOC buffer treatment
After the completion of incubation time the above mixture is centrifuged at 4000 RPM at 2-8°C for 30 minutes to recover pellet. Pellet collected is dissolved in DOC buffer containing 0.3%v/v DOC and 2mM EDTA and 20mM TRIS pH 7.2. (Make up the volume to initial crude volume). Stir the solution for two hours at room temperature.
32% Ethanol precipitation
After completion of incubation period added 6% w/v Sodium Acetate & 0.7% w/v DOC to the above solution the above DOC treatment was carried out under agitation for 10 to 20 minutes adjusting pH at of 6.3 ± 0.1 to this solution added Ethanol to a final concentration of 32% v/v under agitation Adjusting the pH to 6.8± 0.1 using 8M acetic acid, with or without Incubation However incubation is preferred, the solution is precipitated at (2°C - 8°C) for 2 - 4hrs.
AIP04 adsorption
At the end of incubation time, centrifuge the solution at 6000 RPM/30 min at temperature between 2 - 8 °C, discarded pellet, and collected supernatant. To the supernatant solution added A1P04 at a ratio of 1 :8 w/w of polysaccharide and A1P04 (to one gram of polysaccharide add 8g of A1P04 gel) at pH 7.4 kept the solution for 8 -12 hrs under constant stirring conditions at room temperature which forms lipid type solution. The lipid part of the LPS is effectively adsorbed through AIP04 gel under these conditions, which are separated and removed by centrifugation followed by filtration. The resultant product is ready for use.
Gel Precentage Vs Endotoxin results
1 : 1 1 :2 1 :3 1 :4 1 :8 1 : 16 1 :8 1 :8
Specification : < 10IU/ g PRP:Gel w/w
64% Ethanol precipitation
After 24 hours or after a day the above mixture is centrifuged at 6000 RPM at 2-8°C for 30 minutes to recover supernatant. Added sodium acetate to the supernatant to a final concentration of 6% w/v & pH is adjusted to 6.3 ± O. l using 8M Acetic acid, then added ethanol to the solution to a final concentration of 64% v/v under agitation adjusted the pH to 6.8± 0.1 using 8M Acetic Acid, with or without incubation . However incubation is preferred, the solution is precipitated at 2°C - 8°C for 2-4hrs.
300kD Diafiltration followed by 0.2micron filtration
After the end of incubation separate the pellet from above mixture by decanting the supernatant. Dissolved the pellet in WFI (initial volume of crude taken). Clear solution is subjected to diafiltration on 300 kD ULF membrane using 10-20 volumes with WFI. This purified PRP is concentrated and harvested. Then the PRP is filtered through pre sterilized 0.22 μ PES capsule filter and stored in -20°C after sampling.
The product is characterized using sup. l H-NMR data shows that the consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The.1 H-NMR spectrum showed a series of well-resolved signals (protons from the methyl group) for the quantification of functional groups in the polysaccharide. Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide. The purified polysaccharides comply with the WHO specifications for pure polysaccharides.
WHO Specifications for Purified polysaccharides:
Protein Content: - NMT 1% dry weight of the polysaccharide.
Nucleic acid Content: - NMT 1% dry weight of the polysaccharide.
Pyrogen content: - Less then 1 OlU^g of polysaccharide.
EXAMPLE
Taking a thawed PRP kept in the cold room. Diluted the same with PBS for 4 times centrifuged at 6000 rpm per 30 minutes at 2 to 8 degree centigrade, and discarded the pellet. The sup is filtered through 0.45 micron filter and added 10% CTAB to getO.65% final concentration. Incubate at 2 to 8 degree centigrade for 2 hours. Further centrifuged at the rate of 4000 rpm for 30 minutes at 2 to 8 degree, discarded supernatant, precipitate dissolve in 0.5 M Nacl .This solution is filtered through 0.45 micron filter and add ethanol to 72% final concentration incubated at 2 to 8 C for over night.
Centrifuge at 4000 rpm for 30 minutes at 2 to 8 centigrade discarded supernatant ppt dissolve in DOC buffer incubate at RT for 2 hours followed by addition of .0.7% DOC w/v, adjust pH 6.2 add 6% w/v sodium acetate add ethanol to 32% final concentration incubated at 2 to 8 C for 4 hours. Addition of A1P04 at the ratio of 1 :8 (PRP: gel) w/w at pH 7.4 incubated for over night at room temperature. Centrifuge at the rate of 600 rpm, for 30 minutes at 2 to 8 C temperature and discarded the pellet obtained , filter the supernatant through ).22 micron filter add 6% Na -Acetate, adjust pH at 6.1 to 6.3 add ethanol to 62% for final concentration incubate it at 2 to 8° C for 2 to 4 hours. Further centrifuge at 4000 rpm, for 30 minutes and discard supernatant , dissolve the pellet in WFI dia filter on 300 kda membrane with 10 Vol. of WFI , and allow to concentrate and clarify through )0.22 Micron filter and store at -20° C.
Gel Precentage Vs Endotoxin results
1:1 1 :2 1:3 1 :4 1:8 1:16 1:8 1 :8
PRP:Gel
Preparation of Haemophilus influenza - b, Neisseria meningitis such as serotypes
A,C, Y, W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
Claims
1. A process for preparing a pure capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis using aluminum phosphate with alcohol, the said process comprising the following steps, a) precipitating the crude polysaccharide with cetavlon (CTAB - cetyl trimethyl ammonium), taken a crude polysaccharide in the cold room allowed to centrifuge at 6000 RPM for 30 minutes at 2 - 8 °C temperature , after centrifugation discarded the pellet, and collected supernatant solution, to this added 10% Cetavlon to a final concentration of 0.65 %w/v allowed to incubate at room temperature under constant stirring for a period of 2 to 4 hours, b) centrifugated the reaction mixture obtained at step (a) at 1 to 10° C temperature for a period of 15 to 35 minutes to recover pellet, dissolved the pellet in a sodium chloride followed by filtration, c) added 72% Ethanol/or any other organic solvent to the filtrate obtained at step (b) , under constant stirring while maintaining pH 6.8 ± 0.1 using 8 M acetic acid, stored the precipitated solution at room temperature for 2 - 4 hrs/Overnight or without incubation d) after incubation centrifuged at 2-8°C for 30 minutes to recover pellet the said pellets are dissolved in DOC (Sodium Deoxy cholate) buffer containing 0.3%v/v and 2mM EDTA and 20mM TRIS pH 7 stirred the solution for two hours at room temperature, e) added 6% to 8% Sodium acetate and 0.5% to 0.7% DOC (Sodium Deoxy cholate) w/v to the above solution obtained at step (d) under constant stirring condition and organic solvent is added at low concentration followed by with or without incubation, centrifuged to remove contaminants, as pellet. f) added A1P04 while adjusting the pH between 7.0 - 7.4 to the supernatant solution obtained at step (e) and Kept the solution under constant stirring at room temperature to remove the process impurities, g) added 5 to 7%w/v of sodium acetate to the above solution and organic solvent to a make a higher concentration and collected the Polysaccharide as precipitate after incubation followed by centrifugation, h) precipitate collected from the above step (g) is dissolved in WFI and filtered with 10 -20 volumes of WFI using the 300Kd MWCO cassettes the polysaccharide solution is stored at -20°C.
2 A process as claimed in claim 1, wherein the purification of polysaccharide extended to serotypes A,C,Y,W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
3. A process as claimed in claim 1, wherein the DOC (Sodium Deoxy cholate) buffer containing 0.3%w/vand 2mM EDTA (Ethyl dimethyl tetra acetic acid) and 20mM TRIS are used,
4. A process as claimed in claim 2, wherein 6% Sodium acetate and 0.7% DOC w/v , organic solvent is added to a low concentration, organic solvents are selected from ethanol, methanol,
5. A process as claimed in claim 1 and 2, wherein the 0.5% DOC and 32% ethanol pellet are used,
6. A process as claimed in claim 1 , wherein the A1P04 in organic solvent such as
ethanol and / or methanol used for removal of an impurities,
7. A process as claimed in claim 1 , and 4, wherein the impurities such as protein nucleic acid, and endotoxin have been removed,
8. A process as claimed in claim 1, wherein purified polysaccharides are useful for preparation of vaccine for Haemophilus influenza - b, Neisseria meningitis serotypes A,C, Y, W-I35 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
9. A process as claimed in above claims wherein the purified capsular polysaccharides are used for further conjugation with suitable carrier protein to form glyco - conjugate vaccines,
10. A process as claimed in above claims wherein the usage of aluminum phosphate for the purification of capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis serotypes A,C,Y, W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms
1 1. A process for preparing pure capsular polysaccharides of Haemophilus influenza - b, Neisseria meningitis serotypes A,C,Y,W-135 and other similar related capsular polysaccharides produced form both gram negative and gram positive microorganisms using aluminum phosphate with alcohol, such as herein described with reference to an examples and description.
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| IN1640MU2010 | 2010-05-28 |
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| WO2014009971A3 (en) * | 2012-07-07 | 2014-08-28 | Bharat Biotech International Limited | Non-alcoholic vaccine compositions free from animal- origin and process for preparation thereof |
| WO2015128798A1 (en) | 2014-02-25 | 2015-09-03 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | A novel downstream process for purifying polysaccharides |
| WO2017036138A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | A-group neisseria meningitidis capsule crude polysaccharide purification process |
| WO2017036139A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | Preparation process for a-group neisseria meningitidis bacterium capsule crude polysaccharide |
| CN107936128A (en) * | 2017-11-22 | 2018-04-20 | 成都欧林生物科技股份有限公司 | A kind of simple and effective bacterial vaccine purification process |
| KR20180120482A (en) * | 2017-04-27 | 2018-11-06 | 주식회사 유바이오로직스 | Method for manufacturing of Streptococcus pneumonia capsule polysaccharide |
| EP3585427A4 (en) * | 2017-02-24 | 2020-12-30 | Merck Sharp & Dohme Corp. | Methods for improving filterability of polysaccharide-protein conjugate reactions |
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| WO2014009971A3 (en) * | 2012-07-07 | 2014-08-28 | Bharat Biotech International Limited | Non-alcoholic vaccine compositions free from animal- origin and process for preparation thereof |
| CN104487086A (en) * | 2012-07-07 | 2015-04-01 | 巴拉特生物技术国际有限公司 | Non-alcoholic vaccine compositions free from animal- origin and process for preparation thereof |
| CN104487086B (en) * | 2012-07-07 | 2019-08-30 | 巴拉特生物技术国际有限公司 | Non-animal derived nonalcoholic vaccine composition and preparation method thereof |
| WO2015128798A1 (en) | 2014-02-25 | 2015-09-03 | Msd Wellcome Trust Hilleman Laboratories Pvt. Ltd. | A novel downstream process for purifying polysaccharides |
| WO2017036138A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | A-group neisseria meningitidis capsule crude polysaccharide purification process |
| WO2017036139A1 (en) * | 2015-08-31 | 2017-03-09 | 成都欧林生物科技股份有限公司 | Preparation process for a-group neisseria meningitidis bacterium capsule crude polysaccharide |
| EP3585427A4 (en) * | 2017-02-24 | 2020-12-30 | Merck Sharp & Dohme Corp. | Methods for improving filterability of polysaccharide-protein conjugate reactions |
| KR20180120482A (en) * | 2017-04-27 | 2018-11-06 | 주식회사 유바이오로직스 | Method for manufacturing of Streptococcus pneumonia capsule polysaccharide |
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| CN107936128A (en) * | 2017-11-22 | 2018-04-20 | 成都欧林生物科技股份有限公司 | A kind of simple and effective bacterial vaccine purification process |
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| WO2021108792A1 (en) * | 2019-11-29 | 2021-06-03 | Lonza Ltd | Method of purifying polysaccharides |
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