EA041065B1 - METHOD FOR REMOVING IMPURITIES FROM PREPARATIONS BASED ON BACTERIAL CAPSULE POLYSACCHARIDES - Google Patents

Description

The field of technology to which the invention belongs

The present invention generally relates to the field of biotechnology and, in particular, relates to the collection and subsequent processing for purification of bacterial capsular polysaccharides.

State of the art invention

Neisseria meningitidis is the leading cause of infection of the meninges (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord, leading to death and disability worldwide.

Neisseria meningitidis, also known as meningococcus, is a Gram-negative bacterium. According to the structure of the polysaccharide capsule, 13 serogroups of N. meningitidis have been identified, of which 6 serogroups (A, B, C, W, X and Y) have the potential to cause epidemics (see Lee Harrison et al. 2011).

Meningitis occurs in small pockets around the world with seasonal outbreaks of epidemic bacterial meningitis in varying proportions. The greatest burden of meningococcal disease occurs on the African continent, especially in sub-Saharan Africa, also known as the meningitis belt. The Centers for Disease Control and Prevention (CDC) and the World Health Organization estimated that meningococcal disease caused 171,000 deaths worldwide in 2000. In addition, the CDC report confirms the emergence of Neisseria meningitidis as the leading cause of meningitis and sepsis in children and young adults. people in the United States. Thus, Neisseria meningitidis continues to be a public health problem in both developed and developing countries.

In 1996-97 an epidemic caused by Neisseria meningitidis serogroup A has resulted in more than 250,000 cases and more than 25,000 deaths; which further stimulated the global medical community to develop a vaccine that would eliminate the epidemic of group A meningitis in Africa.

In 2010, the meningococcal polysaccharide conjugate vaccine against meningitis A, i.e. Menafrivac, was successfully introduced on the African continent, which led to the almost complete disappearance of epidemics caused by Neisseria meningitidis serogroup A. But with a decrease in the influence of Neisseria meningitidis serogroup A, the influence of other meningococcal serogroups, such as Men-C Men-W, Men-X and Men -Y, began to increase. To counter this dramatic effect, various monovalent and polyvalent vaccines have been developed and brought to market, including the aforementioned serogroup. Although polyvalent conjugate vaccines A, C, Y and W have been licensed since 2005 (Menactra®, Menveo®) for use in children and adults in Canada, the United States of America and Europe, they are available on the market at a very high price, which makes inaccessible to the rest of the world community.

The serogroup A capsule consists of repeating units of O-acetylated (a1-6)-linked N-acetylmannosamine-1-phosphate. Capsular polysaccharides from serogroups B, C, W and Y consist of sialic acid derivatives; serogroups B and C express (α2^8)- and (a2^9)-linked sialic acid homopolymers, and alternating sequences of d-galactose or d-glucose and sialic acid are expressed by serogroups W and Y. Contaminants during broth fermentation interact differently with repeating portions of molecules present on capsular polysaccharides. Therefore, there is a need for a method that exploits these differences to isolate different bacterial capsular polysaccharides from different serogroups.

In accordance with the WHO technical report series on meningococcal conjugate vaccine, isolated polysaccharides must meet the following requirements.

Table 1

Control parameter WHO requirements Mep-S Men W Mep-¥ Polysaccharide content (PS) >3 mg/ml >3 mg/ml >3 mg/ml Protein admixture <1% <5% <5% Nucleic acid impurity <1% <2% <2% Sialic acid content >80% >56% >56% Oacetyl content >1.5 mM per g PS >0.3 mM per g PS >0.3 mM per g PS Content of endotoxin <100 EU per µg PS <100 EU per µg PS <100 EU per µg PS

- 1 041065

Molecular size distribution 75% greater than 0.5 kDa 80% greater than 0.5 kDa 80% greater than 0.5 kDa

Classical methods for purifying bacterial capsular PS used in vaccines involve several selective precipitations with ethanol and/or cationic detergent followed by continuous centrifugation, ultracentrifugation, and phenol deproteinization.

The earliest work to isolate the Neisseria meningitidis polysaccharide from serogroups A, B and C was published by Gotschlich et al. (1969). Gotschlich et al. used the cationic detergent cetavlon (hexadecyltrimethylammonium bromide) to rapidly precipitate negatively charged polysaccharides from an intact culture followed by dissociation of the detergent-polysaccharide complex using 0.9 M CaCl2 extraction and centrifugation, an ethanol precipitation technique was used to remove nucleic acids while proteins were removed by processing polysaccharides with sodium acetate followed by homogenization with chloroform-containing butanol, the alternative use of a hot phenol-water mixture is also reported. The final purification method included the precipitation of polysaccharides with ethanol (4-5 times) and acetone. This method is associated with the inconvenience of using chloroform and large amounts of ethanol. (See T. R. Pato, Master's thesis, Instituto de Quimica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2003, p. 95).

Tanizaki et al. describes a method for purifying Neisseria meningitidis C polysaccharide in which phenol extraction was replaced by proteinase digestion using a mixture of proteinase K, nagars and trypsin; tangential ultrafiltration in hollow fibers with a molecular weight cutoff of 100 kDa instead of ultracentrifugation; followed by vigorous diafiltration using a 100 kDa molecular weight cutoff membrane in 20 mM Tris-HCl buffer containing 0.5% deoxycholate to remove low molecular weight proteins and lipopolysaccharides (LPS). Despite the use of the above modifications, the isolated polysaccharide preparation contained proteins and nucleic acids at 2% (w/w) and 1.5% (w/w), respectively [see Tanizaki et al.; Journal of Microbiological Methods Volume 27, Issue 1, September 1996, Pages 19-23 & Goncalves et al. 2007; formatex; Communicating Current Research and Educational Topics and Trends in Applied Microbiology].

In the work of T.R. Pato et al. (2006) disclosed a modified method for purifying Neisseria meningitidis C polysaccharide, which includes continuous flow culture centrifugation to remove cells; supernatant concentration by tangential filtration (molecular weight cutoff 100 kDa); addition of 0.5% DOC, heating to 55°C for 30 min and tangential filtration (molecular weight cutoff 100 kDa); anion exchange chromatography (source 15Q) and size exclusion chromatography (sepharose CL-4B) [see. T.R. Pato et al./J. Chromatogr. In 832(2006)262-267].

US Pat. No. 7,491,517 B2 discloses the use of CTAB, ethanol, proteinase K, activated charcoal, and gel filtration to remove impurities during purification of the Neisseria meningitidis C polysaccharide. products.

International patent application WO 2017006349 describes the use of zinc acetate/ammonium sulfate/sodium citrate to remove protein contaminants from a harvested N. meningitidis extract. This reference also discloses the use of enzymes such as benzonase, proteinase K or nagarsa to degrade residual proteins and/or nucleic acid materials followed by chromatographic purification.

International patent application WO 2015128798 A1 discloses a method for removing impurities from Neisseria meningitidis C polysaccharide. This method uses incubation at a temperature of 5060°C in the presence of anionic detergents such as sodium deoxycholate or HEPES, deacetylation of the crude polysaccharides using 0.5-1. 5M NaOH at 50° C. for 30 min to 10 h and further purification by diafiltration and hydrophobic interaction chromatography (HIC).

In Tian et al. a method for purifying Neisseria meningitidis serogroups C, W and Y polysaccharides is disclosed, including the use of CTAB, ethanol, DOC, Capto Adhere (multimodal anion exchange chromatography), Capto DEAE (weak anion) and Sephadex G25, where the content of endotoxin is less than 25 EU/mg , protein content less than 10 mg/g, nucleic acid content from 1 to 7 mg/g [see Tian et al. 2013 GE Healthcare; Application note, 29216880AA].

The Gottschlich process, which is a multi-step process, results in a significant loss of product recovery, i.e. about 37%. The use of a chemical such as phenol can lead to undesirable structural changes in the polysaccharide or carrier protein and also results in undesirable phenolic toxic waste.

As described in US 7491517 B2, WO 2017006349 and T.P. Pato et al. (2006) use enzymes that promote the degradation of protein and nucleic acid contaminants, but the removal of enzymes and hydrolyzed material is not an easy task and can lead to loss of the target product. In addition, regulators have restricted the use of animal farms.

- 2,041,065 cops in human products due to the risk of prion contamination. The use of enzymes, in addition to the fact of high cost, leads to more regulatory issues in relation to cGMP, such as the origin of enzymes (from animals or recombinants), differences in enzyme activity between different suppliers and batches, etc.

WO 2017006349 and US 4686102 A disclose the use of ammonium sulfate to precipitate protein and nucleic acid impurities. However, sometimes it also precipitates capsular polysaccharides, resulting in the loss of total polysaccharide.

Sodium deoxycholate (DOC) is a mild detergent and is one of the most commonly used detergents in the purification of polysaccharides. Sodium deoxycholate with a steroid base structure is less denatured and limited in its dissolving power, it destroys endotoxins without affecting the chemical structure; and, therefore, after the removal of sodium deoxycholate, endotoxins regain their biological activity. In addition, DOC-based methods do not work effectively to remove contaminants from polysaccharides, especially polysaccharides containing sialic acid. This may be due to the weak detergent activity of DOC on lipopolysaccharide-protein association formed during subsequent processing, resulting in high levels of endotoxins and protein in the final recovered polysaccharide. In addition, sodium deoxycholate is an animal product, even its residual presence in the final product may result in the product being rejected by regulatory authorities and some religious communities.

Chromatographic techniques such as size exclusion chromatography, ion exchange chromatography, and hydrophobic interaction chromatography have been successfully applied to isolate bacterial polysaccharides with efficient removal of protein and nucleic acid impurities. Despite the successful isolation of the WHO-compliant bacterial polysaccharide, the use of chromatographic methods involves laborious and time-consuming multi-step sample preparation, has problems with scaling, it drastically impairs the recovery of capsular polysaccharides, and thus is not a practical low-cost option for subsequent processing in industrial applications. scales.

Biological leaching accounts for 20-80% of total manufacturing costs (Ansejo and Patrick, 1990), and the development of new post-treatment strategies is essential to reduce manufacturing costs and ensure that the vaccine is available to the entire population through the health system.

Currently, the method used to obtain polysaccharides in their purified forms includes several steps of detergent treatment and differential precipitation of the resulting broth using an organic solvent such as ethanol.

However, the following two main limitations force us to look for an alternative cleaning method.

Currently, the anionic surfactant sodium deoxycholate (DOC) is used as an initial step in the removal of impurities (proteins, nucleic acids and endotoxins). DOC is a bile based animal detergent with a steroid base structure. Due to its bulky structure, it has limited solubility and a lower denaturing capacity for biological macromolecules. In addition, the source of its stable supply with the necessary regulatory certification is limited to one seller/supplier.

The high consumption of ethanol and the inclusion of additional steps (charcoal filtration) make this process laborious, lengthy and expensive.

There remains a significant need for alternative, simple, scalable, and cost-effective methods for purifying bacterial polysaccharides to obtain higher yields of polysaccharides. The present invention relates to a reliable and affordable post-purification method for the isolation of bacterial capsular polysaccharides, wherein said method results in a significant reduction in endotoxin, protein and nucleic acid impurities, thereby providing higher recovery of the polysaccharide as well as maintaining desired O-acetyl levels according to WHO requirements.

The essence of the invention

The present invention relates to an alternative method for purifying bacterial capsular polysaccharides, in particular N. meningitidis capsular polysaccharides.

This method involves the concentration and diafiltration of bacterial biomass by tangential flow filtration with a molecular weight cutoff of 100 kDa, followed by the addition of an anionic detergent and strong alkali to denature proteins, nucleic acids and lipopolysaccharides. Then the biological extract is subjected to centrifugation, diafiltration and precipitation of bacterial polysaccharides using CTAB. The proposed method results in improved polysaccharide yield, is scalable, non-enzymatic, uses cost-effective and fewer purification steps. This method leads to a significant reduction in endotoxin, protein and nucleic acid impurities, which provides more

- 3 041065 more polysaccharide yield and also maintains the desired O-acetyl levels.

List of drawings

Fig. 1 - NMR spectra of the Men-C polysaccharide, FIG. 2 - NMR spectra of the Men-Y polysaccharide, FIG. 3 - NMR spectra of the Men-W polysaccharide, FIG. 4 NMR spectra of Men-A polysaccharide, FIG. 5 - NMR spectra of Men-X polysaccharide.

Description of the invention

In accordance with the general aspect of the invention, one of the bacterial serogroups of interest is grown in a suitable medium and inactivated using formaldehyde or any well-known method from the prior art; the bacterial culture is then post-treated to isolate the purified capsular polysaccharide. Bacteria of interest for isolating the capsular polysaccharide of the present invention are obtained from gram-negative bacteria selected from a genus including, without limitation, Escherichia, Neisseria, Haemophilus, Pseudomonas, etc.; more preferably, the capsular polysaccharide is expressed by the Neisseria meningitidis serogroup. In another aspect of the present invention, the polysaccharide may be derived from the group including 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. , Campylobacter jejuni, Clostridium spp., Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp. , 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 Neisseria meningitidis serogroup A, B, C, D, W135, X, Y, Z, 29E; and H. influenzae type b.

The biological material used during the experiments was as follows.

Polysaccharides have been isolated from

table 2

Name of the organism Strain designation Strain Source Neisseria meningitidis A Μ1027 SynCo Biopartners (Netherlands) Neisseria meningitidis C C11(60E) CBER/FDA, USA Neisseria meningitidis W S877 CBER/FDA, USA Neisseria meningitidis Y M10659 CDC, USA Neisseria meningitidis X M8210 CBER/FDA, USA

Unconjugated carrier protein, ie. CRM197 or TT. CRM197 was obtained from a recombinant strain of Pseudomonas fluorescens CS463-003 (MB101) from Pfenex, USA. TT was derived from Clostridium tetani (Harvard No. 49205) obtained from CRI, National Control Authority, Kazauli, Himachal Pradesh, India. CRI obtained this strain from NVI, The Netherlands.

According to a first embodiment of the invention, the bacterial capsular polysaccharide was purified from the inactivated collection using centrifugation; and the supernatant was subjected to diafiltration using a 100 kDa tangential flow filtration unit. It will be well understood by one skilled in the art that, instead of centrifugation and diafiltration, any other suitable method may be used to concentrate the bacterial capsular polysaccharides. In one preferred embodiment of the invention, the capsular polysaccharide is derived from Neisseria meningitidis serogroups A, C, W, Y, and X.

According to the second embodiment of the invention, the retentate obtained in the first embodiment of the invention was subjected to an anionic surfactant/detergent treatment. The anionic detergent is selected from the group consisting of alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecylsulfonate, sodium s-alkyl sulfates, fatty alcohol polyoxyethylene ether sodium sulfate, sodium oleyl sulfate, sodium N-oleoyl poly(amino acid), sodium alkylbenzenesulfonates, sodium α-olefinsulfonates. , sodium alkylsulfonates, α-sulfomonocarboxylic acid esters, sulfoalkyl fatty acid esters, succinate sulfonate, alkylnaphthalenesulfonates, sodium alkanesulfonates, sodium ligninsulfonate and

- 4 041065 sodium alkylglyceryl ether sulfonates.

Preferably, said anionic surfactant is an alkyl sulfate, more preferably sodium dodecyl sulfate at a final concentration in the range of 0.1 to 4%, more preferably 1%, added to the retentate and stirred at room temperature for 2 hours. Amphipathic nature of SDS makes it a strong chaotropic agent that not only breaks down proteins but also dissolves them.

In another aspect of the second embodiment of the invention, the inactivated harvest can be directly treated with an anionic surfactant and further subjected to concentration of the capsular polysaccharide, resulting in a sufficient reduction in the amount of impurities, which makes the subsequent step of using a cationic detergent optional.

According to the third embodiment of the invention, a strong alkali was added to the mixture obtained in the above embodiment, and the pH was adjusted to 911 with constant stirring at room temperature for 1 hour. Said strong alkali was selected from the group consisting of sodium hydroxide , potassium hydroxide, sodium carbonate, hydroxylamine, triethylamine and lithium hydroxide.

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

In another aspect of the third embodiment of the invention, to the mixture obtained in the second embodiment of the invention, alternatively, instead of alkali, EDTA and sodium acetate were added with constant stirring at room temperature for 1 hour.

According to the fourth embodiment of the invention, the solution obtained in the above embodiment was neutralized (pH 7.0) by adding a mild organic acid. Said soft organic acid is a combination of one or more acids including formic acid, acetic acid, propionic acid, oxalic acid, and the like. Hydrophilic alcohol, preferably ethanol, was added to this neutralized mixture to a final concentration of 30-35% and incubated for 2 hours at room temperature with constant stirring. The hydrophilic alcohol is selected from one of the following alcohols: methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone and t-butyl alcohol, or a combination of two or more of them; and its concentration is <65% or >95%.

In accordance with the fifth embodiment of the invention, excess anionic detergent was removed from the solution. The solution obtained in the above embodiment was subjected to centrifugation and the supernatant was collected. 0.1 M potassium salt was mixed with the supernatant and after its dissolution, the mixture was incubated at a temperature of 2-8°C for >3 hours. The potassium salt was selected from one of the following salts: 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 of them. This step exploits the low solubility of potassium dodecyl sulfate. Upon addition of a potassium salt, preferably KCl, SDS in solution is converted to potassium dodecyl sulfate, being less soluble, it easily precipitates, leading to complete removal of SDS. It will be well understood by one skilled in the art that the concentration of KCl may be varied to obtain the desired result. The protocol mentioned in this embodiment of the invention may be modified in accordance with the requirement of a person skilled in the art.

In another aspect of the fifth embodiment of the invention, excess anionic detergent can be removed from the solution using gel filtration, ethanol precipitation, and ion exchange resins/amberlite columns.

According to the sixth embodiment of the invention, the solution obtained in the above embodiment was subjected to centrifugation, and the supernatant was collected. The supernatant was passed through a 0.2 μm filter and the retentate was diafiltered by tangential flow filtration with a molecular weight cutoff of 100 kDa.

According to the seventh embodiment of the invention, the retentate obtained in the above embodiment was subjected to diafiltration using Tris-HCl buffer at a final concentration of 25 mm. Then cationic detergent was added to a final concentration of 1-2% and incubated at room temperature for 1 h with constant stirring. The cationic detergent(s) are selected from cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrin bromide, or a combination of two or more of them. It will be well understood by one of ordinary skill in the art that the concentration of the cationic detergent may vary from 0.1% to 4% in order to obtain the desired result. Preferably the cationic detergent is CTAB.

According to the eighth embodiment of the invention, the solution obtained in the above embodiment may be subjected to centrifugation, and wasp

- 5 041065 The resulting CTAB polysaccharide is collected and dissolved in 30-64% ethanol. The dissolved mixture may be further subjected to centrifugation to remove undissolved residues.

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

In accordance with the tenth embodiment of the invention, the solution obtained in the above embodiment was subjected to intensive diafiltration with water for injection using 100 kDa tangential flow filtration and passed through a filter with a pore size of 0.2 μm and stored at a temperature -20°C or lower as final weight.

According to an eleventh embodiment of the invention, purified N. meningitidis serogroups C, W, and Y capsular polysaccharide was prepared by the following method.

N. meningitidis serogroups C, W and Y, respectively, were grown in a suitable medium and inactivated using formaldehyde.

To this inactivated collection was added sodium dodecyl sulfate to a final concentration of 1% and stirred at room temperature for 2 hours.

Sodium hydroxide was added to a final concentration of 05-20 mM and the pH was adjusted to 10.5 with constant stirring at room temperature for 1 h.

The solution obtained in the above step was neutralized by adding a mild organic acid, i.e. acetic acid.

Ethanol was added to this neutralized solution to a final concentration of 30-35% and incubated for several hours with constant stirring.

The solution obtained in the above step was centrifuged and the supernatant was collected.

0.1M KCl was added to the supernatant and incubated at 2-8°C for at least 3 hours.

The solution obtained in the above step was subjected to centrifugation and the supernatant was collected. The supernatant was passed through a 0.2 μm filter and the retentate was diafiltered by tangential flow filtration through a 100 kD molecular weight cutoff filter using Tris-HCl buffer.

Tris-HCl buffer was added to the retentate obtained in the above step to a final concentration of 25 mM, followed by the addition of CTAB to a final concentration of 2%; and incubated at room temperature for at least 1 h.

The solution obtained in the above step was centrifuged, the precipitate was collected and dissolved in 30-64% ethanol. The dissolved mixture was then subjected to centrifugation to remove undissolved residues.

NaCl was added to the supernatant obtained in the above step to a final concentration of 0.1-0.3M. The precipitated PS was dissolved in 1 M NaCl, followed by precipitation with 30-64% alcohol.

The solution obtained in the above step was extensively diafiltered against water for injection by tangential flow filtration with a molecular weight cutoff of 100 kDa, passed through a filter with a pore size of 0.2 μm, and stored at -20° C. as the final mass.

According to the twelfth embodiment of the invention, the purification method mentioned in the eleventh embodiment of the invention provides for the production of N. meningitidis serogroup C polysaccharide with a yield of 60-80%, in which the endotoxin content is less than 50 EU/mg, the protein content is less than 0.50% and a nucleic acid content of less than 0.20%, a polysaccharide of N. meningitidis serogroup Y with a yield of 60-80%, in which the endotoxin content is less than 50 EU/mg, the protein content is less than 0.50%, and the nucleic acid content is less than 0.30% , polysaccharide N. meningitidis serogroup W with a yield of 60-80%, in which the content of endotoxin is less than 50 EU/mg, the protein content is less than 0.5% and the content of nucleic acids is less than 0.2%.

According to a thirteenth embodiment of the invention, purified N. meningitidis serogroups A and X capsular polysaccharide was prepared by the following method.

N. meningitidis serogroups A and X, respectively, were grown in a suitable medium and inactivated using formaldehyde.

To this inactivated collection was added sodium dodecyl sulfate to a final concentration of 1% and stirred at room temperature for 2 hours.

To this inactivated collection was added EDTA and sodium acetate to a final concentration of 1% and stirred at room temperature for 2 hours.

Ethanol was added to a final concentration of 30-35% and incubated for 2 h with constant stirring.

- 6 041065

The solution obtained in the above step was centrifuged and the supernatant was collected.

0.1M KCl was added to the supernatant and incubated at 2-8°C for at least 3 hours.

The solution obtained in the above step was subjected to centrifugation and the supernatant was collected. The supernatant was passed through a 0.2 μm filter and the retentate was concentrated and diafiltered by tangential flow filtration through a 100 kDa molecular weight cutoff filter using 25 mM Tris-HCl buffer.

CTAB was added to a final concentration of 1–2% and incubated at room temperature for at least 1 h.

The solution obtained in the above step was centrifuged, the precipitate was collected and dissolved in 30-64% ethanol. The dissolved mixture was then subjected to centrifugation to remove undissolved residues.

NaCl was added to the supernatant obtained in the above step to a final concentration of 0.1-0.3M. The precipitated PS was dissolved in 1 M NaCl, followed by precipitation with 30-64% alcohol.

The solution obtained in the above step was extensively diafiltered against water for injection by tangential flow filtration with a molecular weight cutoff of 100 kDa, passed through a filter with a pore size of 0.2 μm, and stored at -20° C. as the final mass.

According to the fourteenth embodiment of the invention, the purification method mentioned in the thirteenth embodiment of the invention provides for the production of N. meningitidis serogroup A polysaccharide with a yield of 60-80%, in which the endotoxin content is less than 50 EU/mg, the protein content is less than 0.50% and a nucleic acid content of less than 0.20%, a polysaccharide of N. meningitidis serogroup X with a yield of 60-80%, in which the endotoxin content is less than 50 EU/mg, the protein content is less than 0.50%, and the nucleic acid content is less than 0.30% .

According to a fifteenth embodiment of the invention, said purified polysaccharide was conjugated to a carrier protein. It is understood that the carrier protein used for conjugation to the polysaccharides may be any carrier protein known in the art, as required by those skilled in the art. Non-limiting examples of carrier proteins include a carrier protein from the following non-limiting group: CRM 197, diphtheria toxoid, tetanus toxoid, pertussis toxoid, E. coli LT, E. coli ST, Pseudomonas aeruginosa exotoxin A, outer membrane protein complex with (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), pneumococcal surface proteins BVH-3 and BVH-11, Bacillus anthracis protective antigen (PA), detoxified edema-inducing factor (EF), Bacillus anthracis lethal factor (LF), ovalbumin, fisurella hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA) and purified tuberculin protein derivative (PPD).

In another aspect of the fifteenth embodiment of the invention, before conjugation, the polysaccharide purified by this method was brought to a certain size by chemical or mechanical methods, including, without limitation, sonication, microwave treatment, ozonolysis, ionizing radiation treatment, high pressure cell disruption, use of a homogenizer, use of a microfluidizer , sodium acetate treatment, sodium metaperiodate treatment, vacuum heating, etc.

In another aspect of the fifteenth embodiment of the invention, the polysaccharide purified by this method can be conjugated to a carrier protein using reductive amination, cyanylation, carbodiimide conjugation.

In accordance with the sixteenth embodiment of the invention received immunogenic composition. This composition consisted of (a) a conjugate of (i) Neisseria meningitidis serogroup A capsular saccharide and (ii) tetanus toxoid;

(b) a conjugate of (i) Neisseria meningitidis serogroup C capsular saccharide and (ii) CRM197;

(c) a conjugate of (i) Neisseria meningitidis serogroup Y capsular saccharide and (ii) CRM197;

(d) a conjugate of (i) Neisseria meningitidis serogroup W135 capsular saccharide and (ii) CRM197; and (e) a conjugate of (i) Neisseria meningitidis serogroup X capsular saccharide and (ii) tetanus toxoid.

In accordance with the seventeenth embodiment of the invention, the content of endotoxins can be measured using Test for bacterial endotoxins using kinetic turbidimetric analysis (KTA) or testing for pyrogenicity in rabbit; protein content can be measured by the Lowry method; and the nucleic acid content can be measured by spectrophotometry. It is understood that any other suitable method can be used to quantify the content of endotoxin, proteins and nucleic acids.

- 7 041065

In another aspect of the seventeenth embodiment of the invention, the molecular size distribution of the purified Neisseria meningitidis serogroup A, C, W, Y and X polysaccharide may be performed using size exclusion high performance liquid chromatography.

In another aspect of the sixteenth embodiment of the invention, the O-acetyl content can be measured using Hestrin colorimetric analysis.

Examples

Example 1. Obtaining bacterial polysaccharides (the process of accumulation of N. meningitidis serogroup C).

Polysaccharides were obtained using the fermentation process described below.

Fermentation volume: 300 l.

Cleaning in place (CIP), pressure holding test and sterilization in place (SIP) were performed to prepare the fermenter.

After completion of the SIP, the sterile fermentation medium was aseptically transferred to the fermenter.

The composition of the environment.

Table 3

Serogroup C fermentation medium

Ingredient Quantity (g/l) D-glucose monohydrate 10 Sodium chloride 5.8 L-arginine 1 L-serine 1 Calcium chloride 0.028 Iron(II) sulfate heptahydrate 0.01 ammonium chloride 0.3 Dipotassium hydrogen phosphate 4 Yeast extract 5 magnesium chloride 0.4 soy peptone 3 Casaminic acids 5 Monosodium L-Glutamate Monohydrate 10 Ammonium sulfate 1.2 L-cystine 0.3 Culture medium for serogroup C Ingredient Quantity (g/l) D-glucose monohydrate 200 Monosodium L-Glutamate Monohydrate 150 Casaminic acids 4 ammonium chloride 0.2

The level of dissolved oxygen was adjusted to the desired level.

Table 4

The fermentation medium was inoculated with the culture organism. The fermenter operated in the fed-batch mode for 11-14 hours.

After the culture reached the desired OD at 590 nm, the culture in the fermenter was inactivated using formaldehyde.

After completion of the incubation, the temperature was set at 10±5°C, followed by cell separation by centrifugation.

The supernatant was collected and subjected to deep filtration; The primary purified collection was filtered using a filter with a pore size of 0.2 μm and transferred to purification.

- 8 041065

Table 5

results

Parameter Mep-S Total biomass PS yield (mg/l) 703.33 Endotoxin Impurity (EE per mg PS) >500 Protein impurity (%) 109 Nucleic acid impurity (%) 12.3

Example 2 Production of capsular polysaccharides (accumulation process of N. meningitidis serogroups A, W, X and Y).

Using the protocol mentioned in Example 1, N. meningitidis serogroups A, W, X and Y capsular polysaccharide was obtained.

Table 6

Serogroup Y Fermentation Medium

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 Dipotassium hydrogen phosphate 4 Yeast extract 3 magnesium chloride 0.3 soy peptone 3 Casaminic acids 5 Thiamine hydrochloride 0.05 Monosodium L-Glutamate Monohydrate 10 L-tryptophan 0.2

Table 7

Serogroup Y culture medium

Ingredient Quantity (g/l) D-glucose monohydrate 200 Monosodium L-Glutamate Monohydrate 150 L-arginine 1 L-serine 1 soy peptone 10 Yeast extract 16.6

- 9 041065

Table 8

Serogroup W Fermentation Medium

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 Dikal ihydro of osf at 4 Yeast extract 3 magnesium chloride 0.3 soy peptone 3 Casaminic acids 5 Thiamine hydrochloride 0.05 Monosodium L-Glutamate Monohydrate 10

Table 9

Culture medium for serogroup W

Ingredient Quantity (g/l) D-glucose monohydrate 200 Monosodium L-Glutamate Monohydrate 150 L-arginine 4 L-serine 4 soy peptone 4 Yeast extract 4 magnesium chloride 1.6 Calcium chloride 0.2 ammonium chloride 0.8

Table 10

results

Parameter Mep-A Mep-X Men W Men Y crude PS (mg/l) 2540 1024 733 1205 Endotoxin (EE per μg PS) >500 >500 >500 >500 Protein (%) 19.31 18.2 60.5 38 Nucleic acids (%) 1.2 1.16 12 3.3

According to the test results, the initially purified collection contained PS (polysaccharide) in the amount of 1-6 g/l. This initially purified collection was subjected to further purification.

- 10 041065

Example 3 Purification of Capsular Polysaccharides (Isolation and Purification Process of N. meningitidis serogroup C product) using SDS followed by alcohol precipitation.

The crude polysaccharide obtained in accordance with example 1 was mixed with SDS solutions of various concentrations. Ethanol was then added to a final concentration which is approximately 10% below the concentration at which the polysaccharide begins to precipitate. The resulting mixture was further subjected to filtration.

SDS concentrations used: 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

All of the above concentrations of SDS have been shown to be effective in reducing impurities. At SDS concentrations above 4%, no significant differences were observed in the impurity profile.

Optimum reduction of impurities, especially proteins, was observed at a SDS concentration of 1%.

The purified polysaccharide had an endotoxin impurity content of >100 EU/µg polysaccharide, while the WHO limit is <100 EU/µg polysaccharide.

Example 4 Purification of Capsular Polysaccharides (Isolation and Purification Process of N. meningitidis serogroup C product).

Protocol.

Two different experiments were carried out with a fermentation mixture volume of 5 and 300 liters.

The polysaccharides were purified using the purification process described below.

The crude polysaccharide obtained in example 1 was placed in a vessel.

The crude polysaccharide was concentrated 3-6 times by tangential flow filtration with a molecular weight cutoff of 100 kDa.

To this inactivated collection was added sodium dodecyl sulfate to a final concentration of 1% and stirred at room temperature for 2 hours.

Sodium hydroxide was added to a final concentration of 08-20 mM and the pH was adjusted to 10.5 with constant stirring at room temperature for 1 h.

The solution obtained in the above step was neutralized by adding acetic acid.

Ethanol was added to this neutralized solution to a final concentration of 33% and incubated for several hours with constant stirring.

The solution obtained in the above step was centrifuged and the supernatant was collected.

The ethanol concentration was increased to 40%.

0.1M KCl was added to the supernatant and incubated at 2-8°C for at least 3 hours.

The solution obtained in the above step was centrifuged and the supernatant was collected.

The polysaccharide was precipitated by increasing the final concentration of ethanol to 65%.

The polysaccharide precipitate was dissolved in 1M NaCl. The supernatant was passed through a 0.2 μm filter and the retentate was diafiltered by tangential flow filtration through a 100 kD molecular weight cut-off filter using 25 mM Tris-HCl buffer to obtain a purified polysaccharide (step I).

CTAB was added to a final concentration of 2% and incubated at room temperature for 1 h.

The solution obtained in the above step was centrifuged, the precipitate was collected and dissolved in 96% ethanol. The dissolved mixture was then centrifuged to remove undissolved residues.

NaCl was added to the supernatant obtained in the above step to a final concentration of 0.2M. The precipitated PS was dissolved in 1 M NaCl, followed by precipitation of the PS using 65% alcohol.

The solution obtained in the above step was subjected to intensive diafiltration against 0.5 M NaCl, then water for injection by tangential flow filtration through a filter with a molecular weight cut-off of 100 kDa, passed through a filter with a pore size of 0.2 μm and stored at a temperature of -20 °C as final weight.

Table 11

results

N. meningitidis serogroup C Crude polysaccharide Purified Polysaccharide Stage I Purified polysaccharide Stage II Volume 5 l 300 l 5 l 300 l 5 l 300 l

- 11 041065

fermentation PS volume 1 50 0.65 thirty 0.6 18 Concentration PS (mg/ml) 3.87 4.22 3.83 5.62 3.08 7.33 Total Qty PS (gram) 3.87 211 2.48 168 1.84 131.94 Protein impurity (%) 93.79 109.47 0.6 0.52 0.25 0.13 Nucleic acid impurity (%) 7.49 12.32 0.02 0.3 0.02 0.08 Endotoxin impurity (EE per mgc PS) n.p. n.p. <20 <30 <20 40 Extract to stage (%) n.p. n.p. 64.3 79.9 74.23 78.25 Molecule size by HPLC (kDa) n.p. n.p. 434 450 392 376 Oacetyl content Limit: >1.5 mmol/g n.p. n.p. 2.8 2.85 2.64 2.6 Total final recovery (%) n.p. n.p. 64.3 79.9 64 62.5

Note: n.p. = not applicable.

Two different experiments were carried out with the volume of the fermentation mixture 5 and 300 liters.

It was noted that the combined use of an anionic detergent and alkali had a strong effect on the reduction of the impurity profile. The levels of impurities in Purification Step I and Purification Step II are well below the WHO limit values for Mep-C polysaccharide.

At a volume of 5 l and 300 l protein admixture (%) = <1%; nucleic acid impurity (%) = <0.3%; endotoxin impurity (EU per µg PS) = <40 EU per µg PS.

At both volumes and purification step I and purification step II, the recovery of the purified polysaccharide was above 60% compared to the crude sample.

Example 5 Purification of Capsular Polysaccharides (Isolation and Purification Process of N. meningitidis serogroup W and Y product).

Protocol.

Two different experiments were carried out with a fermentation mixture volume of 5 and 300 liters.

The polysaccharides were purified using the purification process described below.

The crude polysaccharide obtained in example 2 was placed in a vessel.

The crude polysaccharide was concentrated 3-6 times by tangential flow filtration with a molecular weight cutoff of 100 kDa.

To this inactivated collection was added sodium dodecyl sulfate to a final concentration of 1% and stirred at room temperature for 2 hours.

Sodium hydroxide was added to a final concentration of 08-20 mM and the pH was adjusted to 10.5 with constant stirring at room temperature for 1 h.

The solution obtained in the above step was neutralized by adding acetic acid.

Ethanol was added to this neutralized solution to a final concentration of 33% and incubated for several hours with constant stirring.

The solution obtained in the above step was centrifuged and the supernatant was collected.

0.1M KCl was added to the supernatant and incubated at 2-8°C for about 8 hours.

The solution obtained in the above step was centrifuged and the supernatant was collected.

- 12 041065

The supernatant was passed through a 0.2 μm filter and the retentate was diafiltered by tangential flow filtration through a 100 kD molecular weight cut-off filter using 25 mM Tris-HCl buffer to obtain a purified polysaccharide (step I).

CTAB was added to a final concentration of 2% and incubated at room temperature for 1 h.

The solution obtained in the above step was centrifuged, the precipitate was collected and dissolved in 96% ethanol. The dissolved mixture was then centrifuged to remove undissolved residues.

NaCl was added to the supernatant obtained in the above step to a final concentration of 0.25M. The precipitated PS was dissolved in 1M NaCl, followed by precipitation of the PS using 65% alcohol.

The solution obtained in the above step was subjected to extensive diafiltration against water for injection by tangential flow filtration through a 100 kDa filter, passed through a filter with a pore size of 0.2 μm, and stored at -20°C as the final mass (stage II).

Results.

Table 12

N. meningitidis serogroup Y Crude polysaccharide Purified Polysaccharide Stage I Purified polysaccharide Stage II Fermentation volume 5 l 300 l 5 l 300 l 5 l 300 l PS volume 1.25 50 0.75 35 0.8 29.5 Concentration PS (mg/ml) 5.67 7.23 7.23 7.88 5.39 8.41 Total Qty PS (gram) 7.08 361.5 5.42 275.8 4.31 248 Protein impurity (%) admixture 38.62 38.03 2.21 1.64 0.16 0.08 nucleic 1.94 3.31 0.69 0.25 0.11 0.71 acids (%) admixture endotoxin n.p. n.p. 50 56 44 45 (EU per MGK PS) Recovery per stage (%) n.p. n.p. 76.50 76.29 79.52 89.95 Size n.p. n.p. 660 620 608 543 molecules by HPLC (kDa) Oacetyl content Limit: >0.3 mmol/g n.p. n.p. 0.85 0.8 0.79 0.75 General final n.p. n.p. 76.50 76.29 60.83 68.62 recovery (%)

- 13 041065

Table 13

N. meningitidis serogroup W Crude polysaccharide Purified Polysaccharide Stage I Purified polysaccharide Stage II Fermentation volume 5 l 300 l 5 l 300 l 5 l 300 l PS volume 1 39 0.8 28.5 0.8 16.5 Concentration PS (mg/ml) 4.26 5.65 3.5 6.01 3.25 8.75 Total Qty PS (gram) 4.26 220 2.8 171 2.6 144 Protein impurity (%) 62.1 60.53 3.4 2.16 <0.21 <0.21 Nucleic acid impurity (%) 7.98 12 0.85 1.16 0.1 0.09 Endotoxin impurity (EE per mgc PS) n.p. n.p. <30 <40 <20 <10 Extract to stage (%) n.p. n.p. 65 77 92 84.2 Molecule size by HPLC (kDa) n.p. n.p. 400 420 395 380 Oacetyl content Limit: >0.3 mmol/g Total final recovery (%) n.p. n.p. n.p. n.p. 1.21 65 1.15 77 1.05 61 0.99 65.4

Two different experiments were carried out with a fermentation mixture volume of 5 and 300 liters.

In comparison, it was observed that the level of impurities in Purification Step I and Purification Step II was well below the WHO limits for Men-Y and Men-W polysaccharide.

It was noted that the combined use of an anionic detergent and alkali had a strong effect on the reduction of the impurity profile. Impurity levels in Purification Step I and Purification Step II are well below the WHO limits for Men-C polysaccharide.

At a volume of 5 l and 300 l protein admixture (%) = <3.5%; nucleic acid impurity (%) = <1.5%; endotoxin impurity (EU per µg PS) = <60 EU per µg PS.

At both volumes and purification step I and purification step II, the recovery of the purified polysaccharide was above 60% compared to the crude sample.

Example 6 Purification of Capsular Polysaccharides (Isolation and Purification Process of N. meningitidis serogroup A and X product).

Protocol.

Two different experiments were carried out with a fermentation mixture volume of 5 and 300 liters.

The polysaccharides were purified using the purification process described below.

The crude polysaccharide obtained in example 2 was placed in a vessel.

The crude polysaccharide was concentrated 3-6 times by tangential flow filtration with a molecular weight cutoff of 100 kDa.

Sodium dodecyl sulfate was added to this inactivated collection to a final concentration of 1% and stirred at room temperature for 2 hours.

Sodium acetate, EDTA, and sodium dodecyl sulfate were added to a final concentration of 6%, 2 mM, and 1%, respectively, with constant stirring at room temperature for 1 h.

The solution obtained in the above step was centrifuged and the supernatant was collected.

0.1M KCl was added to the supernatant and incubated at 2-8°C for about 3 hours.

The solution obtained in the above step was centrifuged and the supernatant was collected.

- 14 041065

The supernatant was passed through a 0.2 μm filter and the retentate was diafiltered by tangential flow filtration through a 100 kDa molecular weight cut-off filter using 25 mM Tris-HCl buffer to obtain a purified polysaccharide (step I).

CTAB was added to a final concentration of 2% and incubated at room temperature for 1 h.

The solution obtained in the above step was centrifuged, the precipitate was collected and dissolved in 96% ethanol. The dissolved mixture was then centrifuged to remove undissolved residues.

NaCl was added to the supernatant obtained in the above step to a final concentration of 0.2M. The precipitated PS was dissolved in 1M NaCl, followed by precipitation of the PS using 65% alcohol.

The solution obtained in the above step was subjected to extensive diafiltration against water for injection by tangential flow filtration through a 100 kDa filter, passed through a filter with a pore size of 0.2 μm, and stored at -20°C as the final mass (stage II).

Results.

Table 14

N. meningitidis serogroup A Crude polysaccharide Purified Polysaccharide Stage I Purified polysaccharide Stage II Fermentation volume 300 l 300 l 300 l PS volume 50 45 thirty Concentration PS (mg/ml) 11.44 6.63 9.75 Total Qty PS (gram) 572 298.35 292.5 Protein impurity (%) 19.31 2.05 0.14 Nucleic acid impurity (%) 1.22 0.12 0.05 Endotoxin impurity (EE per mgc PS) n.p. 29.08 35 Residual content of SDS (ppm) n.p. <5 <5 Extract to stage (%) n.p. 52 98 Molecule size by HPLC (kDa) n.p. 350 306 Oacetyl Content Limit: >2 mmol/g n.p. 2.69 2.65 Total final recovery (%) n.p. 52 51

- 15 041065

Table 15

N. meningitidis serogroup X Crude polysaccharide Purified Polysaccharide Stage I Purified polysaccharide Stage II Fermentation volume 300 l 300 l 300 l PS volume 55 40 thirty Concentration PS (mg/ml) 5.05 5.23 6.21 Total Qty PS (gram) 277.75 209.2 186.30 Protein impurity (%) 18.21 0.6 0.54 Nucleic acid impurity (%) 1.16 0.76 0.14 Endotoxin impurity (EE per mgc PS) n.p. thirty 13.79 Residual chipboard content (ppm) n.p. <5 <5 Extract to stage (%) n.p. 75.3 88.9 Molecule size by HPLC (kDa) n.p. 485 479 Total final recovery (%) n.p. 75.3 67.25

In the comparison, it was observed that the level of impurities in Purification Step I and Purification Step II was well below the WHO limits for Men-A and Men-X polysaccharide.

It was noted that the use of an anionic detergent had a strong effect on the reduction of the impurity profile. The levels of impurities in Purification Step I and Purification Step II are well below the WHO limits for Men-A and Men-X polysaccharide.

At a volume of 300 l, protein impurity (%) = <2.5%; nucleic acid impurity (%) = <1%; endotoxin impurity (EU per µg PS) = <40 EU per µg PS.

At both volumes in purification step I and purification step II, recovery of purified serogroup X polysaccharide was above 60% and serogroup A polysaccharide recovery was above 50% compared to the crude sample.

Example 7 Structural integrity of isolated polysaccharides (PS).

See fig. 1-5 for NMR spectra.

The structural integrity of the isolated N. meningitidis serogroup A, C, W, Y, and X polysaccharides was confirmed by nuclear magnetic resonance (NMR).

The registered NMR spectra were comparable and confirmed their identity as a partially O-acetylated meningococcal polysaccharide of serogroup A, C, W and Y; and N-acetylated serogroup X polysaccharide.

Example 8. Comparative analysis of the purification process based on sodium deoxycholate (DOC) and the claimed method of purification of polysaccharides.

Sodium deoxycholate (DOC) purification method An improved method for meningococcus polysaccharide purification Tanizaki et al. (Conjugate and Polysaccharide Vaccines, Poster 79, http://neisseria.org/ipnc/1996/Neisl996-chap4.pdf) was optimized as follows.

Collection of molecular weight of 100 kDa; treatment with DOC + EDTA + NaOAc + ethanol (40%); centrifugation, collection of supernatant and filtration through a filter with a pore size of 0.2 μm; concentration and diafiltration; treatment with CTAB (3%) at room temperature; centrifugation and collection of precipitated

- 16 041065 CTAB-PS; dissolving the CTAB-PS precipitate in 96% ethanol and precipitating with 0.1M NaCl; dissolving the precipitate in 40% ethanol and adding 1M NaCl (2-8°C); centrifugation and collection of supernatant; coal filtration; precipitation of PS by increasing the concentration of ethanol; dissolving PS granules in water for injection and concentration and diafiltration with water for injection and storage at a temperature of -20°C after filtering through a filter with a pore size of 0.2 μm.

The DOC-based purification method is widely used in the industry for the purification of polysaccharides used as vaccine antigen.

The impurity profile for the polysaccharide obtained by the DOC-based purification process was compared with the claimed method of the present invention.

Results.

Table 16

% final recovery % squirrel % nucleic acids Endotoxin (EE per mgc PS) d DOC 65 <0.3 <0.2 <20 SDS 64 <0.3 <0.2 <20 and d DOC 65 <0.3 <0.2 <20 SDS 62.00 <0.3 <0.2 <20 £th DOC 60 <0.5 <0.5 <30 f SDS 70 <0.5 <0.5 <30 With DOC 60 <0.5 <0.5 <30 SDS 70 <0.5 <0.5 <30 With DOC 65 <0.3 <0.2 <20 sch SDS 70 <0.3 <0.2 <20

The STO based method of the present invention and the DOC based method showed similar results for serogroups A and C, while for serogroups W, Y and X, the STO based method showed improved final recovery compared to the DOC based method.

As can be seen from the above results, the modified SDS method has clear advantages in terms of both ease of operation and a number of other advantages, such as the fact that DOC, which is a component of animal origin, is not compatible with Halal requirements. DOC is produced and supplied by a single supplier worldwide, while SDS is a Halal certified synthetic detergent and has several suppliers available worldwide. DOC breaks down endotoxins without affecting the chemical composition, and after removing this detergent, they can restore their biological activity, while SDS, due to its amphipathic nature and higher aggregation number, denatures and dissolves proteins well, and also irreversibly destroys endotoxins to their monomer units.

When using SDS, there is a reduced consumption of chemicals and consumables (ethanol, ultrafilters, carbon filters, etc.), which is a clear advantage. In addition, the use of SDS as a replacement is non-invasive to the structural integrity of the polysaccharides.

Example 9

An immunogenic composition as shown below was prepared and tested for immunogenicity.

The composition consisted of (a) a conjugate of (i) Neisseria meningitidis serogroup A capsular saccharide and (ii) tetanus toxoid;

(b) a conjugate of (i) Neisseria meningitidis serogroup C capsular saccharide and (ii) CRM197;

(c) a conjugate of (i) Neisseria meningitidis serogroup Y capsular saccharide and (ii) CRM197;

(d) a conjugate of (i) Neisseria meningitidis serogroup W135 capsular saccharide and (ii) CRM197; and (e) a conjugate of (i) Neisseria meningitidis serogroup X capsular saccharide and (ii) tetanus toxoid.

Results.

The immunogenic composition was found to be immunogenic.

Benefits of the Invention

This method leads to a significant reduction in endotoxin, protein and nucleic acid impurities, thereby providing a higher recovery of capsular polysaccharide with the desired

-

Claims (43)

O-acetyl level. As can be seen from the above results, the claimed method has clear advantages in terms of ease of operation, as well as a number of other advantages, such as a regulatory compliance problem: DOC is an animal-derived component and is not Halal compliant. It is produced and supplied by a single supplier worldwide, while SDS is a Halal certified synthetic detergent and also has several suppliers available worldwide; functional problem: DOC breaks down endotoxins without affecting their chemical composition, and after removing this detergent, they can restore their biological activity, while SDS, due to its amphipathic nature and higher aggregation number, denatures and dissolves proteins, and also irreversibly destroys endotoxins to their monomeric units. When using SDS, there is a reduced consumption of chemicals and consumables (ethanol, ultrafilters, carbon filters, etc.), which is a clear advantage. The method described in the present invention leads to a significant reduction in other cellular impurities. In addition, this method provides efficient removal of reagents used during primary and subsequent processing. In addition, the method described in the present invention is non-invasive to the structural integrity of the polysaccharides, allowing them to retain their immunogenicity. In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be understood that illustrative embodiments of the invention are only preferred examples of the invention and should not be construed as limiting the scope of the invention. CLAIM 1. A method for isolating a polysaccharide from Neisseria Meningitidis selected from serotype C, serotype W and serotype Y, which includes the following steps: a) mixing the crude polysaccharide solution with sodium dodecyl sulfate (SDS); b) adding sodium hydroxide (NaOH) to the solution; c) neutralizing the pH of the solution; d) alcohol precipitation of the solution followed by centrifugation and retention of the supernatant; e) removing sodium dodecyl sulfate present in the supernatant; f) concentration and diafiltration of the polysaccharide; where the obtained purified polysaccharide has a degree of extraction of 60-80%. 2. The method according to claim 1, where the method further includes the following steps: g) mixing the polysaccharide solution with the cationic detergent; h) centrifuging the solution and collecting the pellet, and i) dissolving the precipitate in alcohol followed by precipitation of the polysaccharides using salt. 3. The method according to claim 1, in which a) the purified polysaccharide from Neisseria Meningitidis serotype C has O-acetylation levels >1.5 mmol/g, protein contaminant <1%, nucleic acid contaminant <1%, and endotoxin contaminant <50 EU/μg; b) the purified polysaccharide from Neisseria Meningitidis serotype Y has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <60 EU/µg; And c) Purified polysaccharide from Neisseria Meningitidis serotype W has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <50 EU/µg. 4. The method according to claim 3, where the final concentration of sodium dodecyl sulfate (SDS) in the solution is in the range from 0.1 to 4.0%, preferably less than 1%. 5. The method of claim 1 wherein the final concentration of sodium hydroxide in solution is in the range of 5 to 50 mM, preferably in the range of 5 to 20 mM, and most preferably in the range of 8 to 15 mM. 6. The method according to claim 1, wherein the pH of the solution after the addition of sodium hydroxide is in the pH range from 9 to 11, preferably the pH is 10.5. 7. The method of claim 1 wherein the solution is neutralized (pH 7.0) by adding a mild organic acid. 8. The method according to claim 7, where the soft organic acid is formic acid, acetic acid, propionic acid, oxalic acid; preferably acetic acid. 9. The method according to claim 1, where the removal of sodium dodecyl sulfate is carried out using at least one or more of the following operations: treatment with a potassium salt, gel filtration, treatment with ethanol and the use of ion exchange resins/amberlite columns. - 18 041065 10. The method according to claim 1, where the removal of sodium dodecyl sulfate is carried out by precipitation using a potassium salt. 11. The method of claim 10 wherein the potassium salt is 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 of them. . 12. The method of claim 11 wherein the potassium salt is potassium chloride. 13. The method of claim 10 wherein the final concentration of the potassium salt in the solution is greater than or equal to the concentration at which sodium dodecyl sulfate is sufficiently precipitated. 14. The method of claim 10, wherein the final concentration of the potassium salt in the solution is less than 300 mM, preferably 100 mM. 15. The method of claim 1 wherein the polysaccharide is a polymer of sialic acid residues. 16. The method of claim 2, wherein the cationic detergent (a) is a cetyltrimethylammonium salt, a tetrabutylammonium salt, a myristyltrimethylammonium salt, and hexadimethrin bromide, or a combination of two or more of them. 17. The method of claim 2 wherein the cationic detergent (a) is cetyltrimethylammonium bromide (CTAB). 18. The method according to claim 17, wherein the concentration of cationic detergent (b) is less than 3%, preferably 1%. 19. The method according to claims 1 and 2, wherein the alcohol is a hydrophilic alcohol. 20. The method according to claim 19, where the hydrophilic alcohol is methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone and tert-butyl alcohol, or a combination of two or more of them; and its concentration is <65% or >95%. 21. The method according to claims 1 and 2, where the alcohol is ethanol. 22. The method of claim 1, wherein the concentration and diafiltration of the polysaccharide is carried out by tangential flow filtration using a (MWCO) membrane with a molecular weight cutoff of 100 kDa. 23. The method of claim 2, wherein the salt used for precipitation is sodium chloride, ammonium chloride, potassium chloride, or a combination of two or more of these. 24. The method of claim 2 wherein the salt used for precipitation is sodium chloride. 25. The method according to claim 2, where the salt concentration is in the range from 0.25 to 2M. 26. A method for isolating a polysaccharide from Neisseria Meningitidis selected from serotype C, serotype W and serotype Y, comprising the following steps: a) adding sodium dodecyl sulfate (SDS) to the crude polysaccharide solution; b) adding EDTA and sodium acetate to the solution; c) alcohol precipitation of the solution, followed by centrifugation and retention of the supernatant; d) removing sodium dodecyl sulfate present in the supernatant; e) concentration and diafiltration of the polysaccharide; where the obtained purified polysaccharide has a degree of extraction of 60-80%. 27. The method of claim 26, wherein the method further comprises the steps of: a) mixing the bacterial polysaccharide solution with the cationic detergent; b) centrifuging the solution and collecting the precipitate; c) dissolving the precipitate in alcohol followed by precipitation of the polysaccharides using salt. 28. The method according to claim 26, in which a) purified polysaccharide from Neisseria Meningitidis serotype A has O-acetylation levels >2 mmol/g, protein contaminant <3%, nucleic acid contaminant <1% and endotoxin contaminant <50 EU/μg; and b) The purified polysaccharide from Neisseria Meningitidis serotype X has <1% protein contaminant, <1% nucleic acid contaminant, and <50 EU/μg endotoxin contaminant. 29. The method of claim 26, wherein the final concentration of sodium dodecyl sulfate (SDS) is in the range of 0.1 to 4.0%, preferably less than 1%. 30. The method of claim 26, wherein the removal of sodium dodecyl sulfate (SDS) is carried out using at least one or more of the following operations: potassium salt treatment, gel filtration, ethanol treatment, and the use of ion exchange resins/amberlite columns. 31. The method according to claim 26, wherein the removal of sodium dodecyl sulfate (SDS) is carried out by precipitation using a potassium salt. 32. The method of claim 31, wherein the final concentration of the potassium salt in the solution is greater than or equal to the concentration at which sodium dodecyl sulfate (SDS) is sufficiently precipitated. 33. The method according to claim 31, where the potassium salt is 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 of them . 34. The method according to claim 27, where the cationic detergent (a) is a salt of cetyltrimethylammonium, - 19 041065 tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrin bromide or a combination of two or more of them. 35. The method of claim 27 wherein the cationic detergent (a) is cetyltrimethylammonium bromide (CTAB). 36. The method of claim 35 wherein the final concentration of CTAB is in the range of 0.5 to 3.0%, preferably less than 1%. 37. The polysaccharide obtained by the method according to any one of claims 1 or 26, while the polysaccharide is: a) the purified polysaccharide from Neisseria Meningitidis serotype C has O-acetylation levels >1.5 mmol/g, protein contaminant <1%, nucleic acid contaminant <1%, and endotoxin contaminant <50 EU/µg; or b) the purified polysaccharide from Neisseria Meningitidis serotype Y has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <60 EU/µg; or c) Purified polysaccharide from Neisseria Meningitidis serotype W has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <50 EU/μg. 38. The polysaccharide obtained by the method of claim 1 or 26 is a capsular polysaccharide, a subcapsular polysaccharide or an exopolysaccharide. 39. The polysaccharide according to claim 1 or 26, where the polysaccharide is brought to a certain size by chemical or mechanical means. 40. A vaccine containing a polysaccharide antigen obtained by the method according to claim 1 or 26, while the polysaccharide antigen is: a) the purified polysaccharide from Neisseria Meningitidis serotype C has O-acetylation levels >1.5 mmol/g, protein contaminant <1%, nucleic acid contaminant <1%, and endotoxin contaminant <50 EU/µg; or b) the purified polysaccharide from Neisseria Meningitidis serotype Y has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <60 EU/µg; or c) Purified polysaccharide from Neisseria Meningitidis serotype W has O-acetylation levels >0.3 mmol/g, protein contaminant <5%, nucleic acid contaminant <2% and endotoxin contaminant <50 EU/μg. 41. A conjugate of a polysaccharide obtained by the method of claim 1 or 26 with a carrier protein, wherein the polysaccharide is conjugated to a carrier protein, wherein the carrier protein is CRM197, pneumococcal surface adhesin A (PsaA), diphtheria toxoid (DT), tetanus toxoid (TT), tetanus toxoid C fragment, pertussis toxoid, H. influenzae protein D, E. coli LT, E. coli ST, Pseudomonas aeruginosa exotoxin A, outer membrane protein complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3, BVH-11, Bacillus anthracis protective antigen (PA), detoxified edema-inducing factor (EF), lethal factor (LF) Bacillus anthracis, ovalbumin, fisurella hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified tuberculin protein derivative (PPD); in particular CRM197, tetanus toxoid, diphtheria toxoid and/or PsaA. 42. A polysaccharide conjugate according to claim 41 conjugated to a carrier protein using reductive amination, cyanylation, carbodiimide conjugation. 43. An immunogenic composition containing at least one of the conjugates of claim 41; at least one polysaccharide protein conjugate; sucrose at a concentration of 3% wt./about. and sodium citrate at a concentration of 0.5% w/v; where the polysaccharide is purified by the method according to claim 1 or 26. -