A, C, Y and W135 group meningococcus polysaccharide chromatography method

Abstract

The invention provides a method for chromatography of meningococcus polysaccharides in groups A, C, Y and W135. Specifically, the invention provides a purification method of meningococcal polysaccharide, which comprises the following steps: 1) respectively pretreating meningococcal polysaccharide A, meningococcal polysaccharide C, meningococcal polysaccharide Y and meningococcal polysaccharide W135 by using 0.5-2% sodium deoxycholate; 2) utilizing a Rigose MMA chromatographic column and a Rigose DEAE chromatographic column which are connected in series, and purifying the polysaccharide pretreated in the step 1); (3) carrying out ultrafiltration desalination on the polysaccharide in the step (2) by using 10-100mM of a phosphoric acid buffer solution to obtain purified refined polysaccharide; wherein the meningococcus polysaccharide is meningococcus group A polysaccharide, meningococcus group C polysaccharide, meningococcus group Y polysaccharide or meningococcus group W135 polysaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide preparation, and more specifically, the invention relates to a method for chromatographic separation of group A, C, Y and W135 meningococcal polysaccharides. Background Art

[0002] Epidemic cerebrospinal meningitis, referred to as meningitis, is an acute respiratory infectious disease caused by Neisseria meningitidis. The pathogenic bacteria of meningitis include group A, group C, group Y, and group W135 meningococci. Extracting the bacterial outer capsule polysaccharide of a specific strain of meningococci and preparing it into a vaccine can be used to prevent invasive infection by the strain of meningococci.

[0003] Capsular polysaccharides are macromolecules, and separation and purification are more complicated than monosaccharides or small molecule oligosaccharides. In the separation and purification of capsular polysaccharides, impurities such as nucleic acids, proteins, and endotoxins need to be removed. At present, the preparation process of meningococcal polysaccharides adopts steps such as phenol extraction and ethanol precipitation. Due to the use of multi-step ethanol precipitation and phenol extraction, the process is relatively complicated, the manual operation is highly different, the batch yield is not completely consistent, and the process amplification is limited. In addition, phenol is highly toxic, and phenol residues will affect product quality and the human body. In addition, the selection of meningococcal polysaccharide chromatography medium will also affect the quality of the product.

[0004] Therefore, there is an urgent need in the art to provide an improved method for chromatography of group A, C, Y, and W135 meningococcal polysaccharides. Summary of the invention

[0005] The object of the present invention is to provide a method for chromatographic separation of group A, C, Y and W135 meningococcal polysaccharides.

[0006] In a first aspect of the present invention, a method for purifying meningococcal polysaccharide is provided, comprising:

[0007] 1) pre-treating meningococcal polysaccharide with 0.5-2% sodium deoxycholate;

[0008] 2) purifying the polysaccharide pretreated in step 1) by connecting a Rigose MMA chromatography column and a Rigose DEAE chromatography column in series;

[0009] 3) using a phosphate buffer to ultrafilter and desalt the polysaccharide in step 2) to obtain purified polysaccharide;

[0010] Wherein, the meningococcal polysaccharide is meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, or meningococcal group W135 polysaccharide.

[0011] In one or more embodiments, in step 1), the content of sodium deoxycholate is 0.5-2%, preferably 0.6-1.9%, 0.7-1.8%, 0.8-1.7%, 0.9-1.6% or 1.0-1.5%, wherein the content is expressed as mass percentage.

[0012] In one or more embodiments, in step 1), the treatment time of sodium deoxycholate is 1 to 15 hours, preferably 2 to 14 hours, 3 to 13 hours, 3 to 12 hours, 5 to 12 hours, 6 to 12 hours, 7 to 12 hours, 8 to 12 hours or 9 to 12 hours.

[0013] In one or more embodiments, in step 1), the sodium deoxycholate is formulated in a buffer system, and the buffer system further comprises 1-100 mM Tris-HCl buffer, preferably 2-90 mM, 3-80 mM, 5-50 mM, 8-40 mM, 10-30 mM or 15-25 mM; the pH value of the buffer solution is 6.0-9.0, preferably 6.5-8.5, 6.5-8.0, 7.0-8.5 or 7.5-8.0.

[0014] In one or more embodiments, the step 2) further comprises: balancing the chromatography column with a buffer.

[0015] In one or more embodiments, the step 2) further comprises: pre-balancing the chromatography column with a buffer solution that does not contain sodium deoxycholate, and then balancing the chromatography column with a buffer solution that contains 0.5-2% sodium deoxycholate.

[0016] In one or more embodiments, in step 2), the buffer solution is 1-100 mM Tris-HCl buffer, preferably 2-90 mM, 3-80 mM, 5-50 mM, 8-40 mM, 10-30 mM or 15-25 mM; the pH value of the buffer solution is 6.0-9.0, preferably 6.5-8.5, 6.5-8.0, 7.0-8.5 or 7.5-8.0.

[0017] In one or more embodiments, in step 3), the ultrafiltration desalination comprises: ultrafiltration of the polysaccharide in step 2) using a phosphate buffer, and then ultrafiltration elution with water for injection to remove the phosphate buffer.

[0018] In one or more embodiments, in step 3), the concentration of the phosphate buffer is 10-100 mM, preferably 20-90 mM, 30-80 mM, 40-70 mM, 40-60 mM or 45-55 mM.

[0019] In one or more embodiments, the step 3) further comprises: freeze-drying the polysaccharide solution after ultrafiltration and desalination, thereby obtaining a purified and refined polysaccharide in a solid form.

[0020] In one or more embodiments, in step 1), the crude meningococcal polysaccharide is a crude meningococcal polysaccharide obtained by separation from fermentation broth of meningococcal pathogenic bacteria.

[0021] In one or more embodiments, the meningitis-causing bacteria include or are Neisseria meningitidis.

[0022] In one or more embodiments, the meningitis pathogen includes or is selected from: Neisseria meningitidis serogroups A, C, Y, and W135.

[0023] In one or more embodiments, the step of isolating crude polysaccharides using fermentation broth of meningococcal pathogenic bacteria includes: fermenting and culturing group A, group C, group Y or group W135 meningococcal pathogenic bacteria, inactivating the fermentation broth and then centrifuging it, adding a cationic detergent and a polysaccharide precipitant to the centrifugal supernatant, and isolating and obtaining crude meningococcal polysaccharides.

[0024] In one or more embodiments, the cationic detergent is CTAB and / or the polysaccharide precipitating agent is ethanol.

[0025] In one or more embodiments, after adding CTAB and ethanol, the mass percentage of CTAB in the solution is 0.1±0.05%, and the volume percentage of ethanol is 75±1%.

[0026] In a second aspect of the present invention, a meningococcal polysaccharide is provided, which is obtained by the purification method described in any embodiment of the present invention; wherein the meningococcal polysaccharide is meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, or meningococcal group W135 polysaccharide.

[0027] In one or more embodiments, the meningococcal polysaccharide has one, more or all of the following characteristics:

[0028] Protein content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2 mg / g;

[0029] Nucleic acid content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2.5 mg / g;

[0030] The phosphorus content of group A polysaccharide is >80 mg / g, preferably >82 mg / g, >83 mg / g, >84 mg / g, >85 mg / g, >86 mg / g or >87 mg / g;

[0031] The sialic acid content of group C polysaccharide is >800 mg / g, preferably >802 mg / g, >803 mg / g, >804 mg / g, >805 mg / g, >806 mg / g or >807 mg / g;

[0032] The sialic acid content of group Y polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g or >570 mg / g;

[0033] The sialic acid content of the W135 group polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g, >570 mg / g, >572 mg / g, >575 mg / g, >577 mg / g, >578 mg / g or >579 mg / g;

[0034] Group A O-acetyl content ≥2.0mmol / g, preferably ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g;

[0035] Group C O-acetyl content ≥1.5mmol / g, preferably ≥1.7mmol / g, ≥1.8mmol / g, ≥1.9mmol / g, ≥2.0mmol / g, ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g;

[0036] The O-acetyl content of group Y is ≥0.3 mmol / g, preferably ≥0.4 mmol / g, ≥0.5 mmol / g, ≥0.6 mmol / g, ≥0.8 mmol / g, ≥1.0 mmol / g, ≥1.1 mmol / g or ≥1.2 mmol / g;

[0037] W135 group O-acetyl content ≥ 0.3mmol / g;

[0038] Endotoxin is <12.5 EU / μg, preferably <12 EU / μg, <11 EU / μg, <10 EU / μg, <9 EU / μg, <8 EU / μg, <7 EU / μg or <6 EU / μg.

[0039] In a third aspect of the present invention, a conjugate is provided, comprising one or more selected from the following:

[0040] (1) Meningococcal group A polysaccharide and a carrier protein conjugated thereto,

[0041] (2) Meningococcal group C polysaccharide and carrier protein conjugated thereto,

[0042] (3) Meningococcal group Y polysaccharide and carrier protein conjugated thereto,

[0043] (4) Meningococcal group W135 polysaccharide and carrier protein conjugated thereto;

[0044] Among them, the meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, and meningococcal group W135 polysaccharide are the meningococcal polysaccharides described in any embodiment of the present invention, or are obtained by the purification method described in any embodiment of the present invention.

[0045] In one or more embodiments, the carrier protein is a protein or a fragment thereof that is acceptable to an individual and can induce an immune cell-dependent response.

[0046] In one or more embodiments, the carrier protein has at least one free amino group for conjugation with a polysaccharide.

[0047] In one or more embodiments, the carrier protein comprises: tetanus toxoid, cholera toxin, diphtheria toxoid, toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including E. coli.

[0048] In one or more embodiments, the tetanus toxoid, cholera toxin, diphtheria toxoid, toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including Escherichia coli comprise mutants thereof.

[0049] In one or more preferred embodiments, the diphtheria toxoid comprises a diphtheria toxoid mutant.

[0050] In a fourth aspect of the present invention, a composition is provided, comprising: the meningococcal polysaccharide described in any embodiment of the present invention, and / or the conjugate described in any embodiment of the present invention.

[0051] In one or more embodiments, the composition further comprises an adjuvant.

[0052] In one or more embodiments, the adjuvant comprises one or more selected from the group consisting of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid A, QS21, CpG, stearoyl tyrosine, and Freund's adjuvant.

[0053] In the fifth aspect of the present invention, there is provided the use of the meningococcal polysaccharide described in any embodiment of the present invention, or the conjugate described in any embodiment of the present invention, or the composition described in any embodiment of the present invention in the preparation of a medicament for providing protection against infection by bacterial meningitis pathogens.

[0054] In one or more embodiments, the medicament is a vaccine.

[0055] In one or more embodiments, the meningitis-causing bacteria include or are Neisseria meningitidis.

[0056] In one or more embodiments, the meningitis pathogen includes or is selected from: Neisseria meningitidis serogroups A, C, Y, and W135.

[0057] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 , a chromatographic purification map of the first batch of novel meningococcal group A crude polysaccharides.

[0059] Figure 2 , the chromatographic purification of the second batch of crude polysaccharides of novel meningococcal group A.

[0060] Figure 3 , the chromatographic purification of the third batch of crude polysaccharides of novel meningococcal group A.

[0061] Figure 4 , a chromatographic purification map of the first batch of novel meningococcal group C crude polysaccharides.

[0062] Figure 5 , the chromatographic purification of the second batch of crude polysaccharides of novel meningococcal group C.

[0063] Figure 6 , the chromatographic purification of the third batch of crude polysaccharides of novel meningococcal group C.

[0064] Figure 7 , chromatographic purification of the first batch of crude polysaccharides of novel meningococcal group Y.

[0065] Figure 8 , the chromatographic purification of the second batch of crude polysaccharides of novel meningococcal group Y.

[0066] Fig. 9 , the chromatographic purification of the third batch of crude polysaccharides of novel meningococcal group Y.

[0067] Fig.10 , a chromatographic purification diagram of the first batch of crude polysaccharides of the novel meningococcal W135 group.

[0068] Fig.11 , the chromatographic purification of the second batch of crude polysaccharides of the novel meningococcal W135 group.

[0069] Fig.12 , the chromatographic purification of the third batch of crude polysaccharides of novel meningococcal W135 group. DETAILED DESCRIPTION

[0070] After in-depth research, the inventors have established a new chromatography purification process for crude polysaccharides of group A, group C, group Y, and group W135 of meningococci: after the crude polysaccharides of group A, group C, group Y, and group W135 are dissolved in Tris-HCl buffer, they are diluted with an equal volume of Tris-HCl buffer containing sodium deoxycholate (DOC), and the polysaccharides are separated from other impurities by one-step chromatography using Rigose MMA and Rigose DEAE chromatography media in series, and the polysaccharides are separated from other impurities by a flow-through mode, and the harvested liquid is subjected to ultrafiltration, filtration, and freeze-drying with 50mM phosphate buffer (PB) and sterile water for injection to obtain refined polysaccharides, and the polysaccharide stock solution is tested according to the methods and standards of the "Chinese Pharmacopoeia". The results show that the four groups of meningococcal polysaccharides can be completely separated from proteins and nucleic acids, and the detection indicators of the refined polysaccharides after chromatography purification meet the standards of the "Chinese Pharmacopoeia", and the recovery rate of polysaccharides in each group is higher than 80%.

[0071] the term

[0072] As used herein, the term "meningococcus" or "meningitis pathogenic bacteria" refers to bacteria that can cause bacterial meningitis, including but not limited to Neisseria meningitidis, such as 1, 2, 3 or 4 of Neisseria meningitidis A, C, Y, W135 serogroups. In some embodiments, the polysaccharide molecular weight of A, C, Y, W135 serogroups is between 40-400 kD. In some embodiments, the bacterial meningitis pathogenic bacteria include Neisseria meningitidis A, C, Y and / or W135 serogroups.

[0073] As used herein, the term "purification" refers to the process of removing non-polysaccharide components from crude polysaccharides extracted from meningococci to obtain refined polysaccharides. The non-polysaccharide components include, but are not limited to, proteins, nucleic acids, phosphoric acid, sialic acid, O-acetyl groups, and endotoxins. Herein, the purification can be performed using a chromatographic method.

[0074] As used herein, the term "polysaccharide recovery rate" refers to the percentage of the effective components of the refined polysaccharide after purification to the effective components of the crude polysaccharide before purification.

[0075] Method for purifying polysaccharides of meningococcal group A, group C, group Y and group W135

[0076] The present invention provides a method for purifying meningococcal polysaccharide, comprising:

[0077] 1) using 0.5-2% sodium deoxycholate to pretreat meningococcal group A, group C, group Y, and group W135 polysaccharides respectively;

[0078] 2) purifying the polysaccharide pretreated in step 1) by connecting a Rigose MMA chromatography column and a Rigose DEAE chromatography column in series;

[0079] 3) using 10-100 mM phosphate buffer to ultrafilter and desalt the polysaccharide in step 2) to obtain purified polysaccharide;

[0080] Wherein, the meningococcal polysaccharide is meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, or meningococcal group W135 polysaccharide.

[0081] In step 1), the sodium deoxycholate is an anionic detergent that can perform preliminary separation of polysaccharides from proteins, nucleic acids and endotoxins. In step 1), the content of sodium deoxycholate is 0.5-2%, such as 0.6-1.9%, 0.7-1.8%, 0.8-1.7%, 0.9-1.6% or 1.0-1.5%, wherein the content is expressed as a mass percentage. The treatment time of sodium deoxycholate can be 1-15 hours, such as 2-14 hours, 3-13 hours, 3-12 hours, 5-12 hours, 6-12 hours, 7-12 hours, 8-12 hours or 9-12 hours. Typically, the 0.5-1.5% sodium deoxycholate is formulated in a buffer system, the buffer system further comprising a Tris-HCl buffer, the concentration of which may be 1-100 mM, preferably 2-90 mM, 3-80 mM, 5-50 mM, 8-40 mM, 10-30 mM or 15-25 mM, such as 20 mM. The pH value of the buffer solution is typically 6.0-9.0, such as 6.5-8.5, 6.5-8.0, 7.0-8.5 or 7.5-8.0.

[0082] In step 1), the polysaccharide of group A, group C, group Y, and group W135 of meningococci is a crude polysaccharide. The crude polysaccharide can be a polysaccharide obtained from group A, group C, group Y, or group W135 of Neisseria meningitidis. For example, group A, group C, group Y, or group W135 of Neisseria meningitidis is fermented and cultured, the fermentation broth is inactivated and then centrifuged (for example, continuous flow centrifugation or disc centrifugation), and a cationic detergent (CTAB) and a polysaccharide precipitant (ethanol) are added to the centrifugal supernatant to obtain the crude polysaccharide.

[0083] In step 2), when the Rigose MMA chromatography column and the Rigose DEAE chromatography column are connected in series, since Rigose MMA is a composite mode chromatography medium, it has both ion exchange mode and hydrophobic mode, and Rigose DEAE is a weak anion exchange medium. The two are connected in series, and can bind non-polysaccharide components through electrostatic action, and can also bind non-polysaccharide components through hydrophobic action, and can well remove non-polysaccharide components. Moreover, under the DOC buffer system, a large number of deoxycholates occupy the binding sites on the chromatography column, forming competitive inhibition with polysaccharides, so that non-polysaccharide components such as proteins, nucleic acids and endotoxins with strong electrostatic effects are bound to the chromatography column, and polysaccharides are eluted through the flow-through mode, thereby separating and purifying polysaccharides well.

[0084] In some preferred embodiments, in step 2), the Rigose MMA chromatography column and the Rigose DEAE chromatography column are domestic chromatography columns. Compared with the method using imported chromatography media, i.e., Capto Adhere chromatography column, Capto DEAE chromatography column and Sephodex G25 chromatography column, the cost of using Rigose MMA chromatography column and Rigose DEAE chromatography column is lower, and can produce a more excellent technical effect of purifying and refining polysaccharides.

[0085] The step 2) may also include: balancing the chromatography column with a buffer. The balancing of the chromatography column with a buffer may be performed once, twice, three times or more. Exemplarily, the chromatography column may be pre-balanced with a buffer solution that does not contain sodium deoxycholate, and then the chromatography column may be balanced with a buffer solution containing 0.5 to 1.5% (mass percentage) sodium deoxycholate. The buffer solution may be a Tris-HCl buffer, and its concentration may be 1 to 100 mM, preferably 2 to 90 mM, 3 to 80 mM, 5 to 50 mM, 8 to 40 mM, 10 to 30 mM or 15 to 25 mM, for example 20 mM. The pH value of the buffer solution is generally 6.0 to 9.0, for example 6.5 to 8.5, 6.5 to 8.0, 7.0 to 8.5 or 7.5 to 8.0.

[0086] In step 3), the concentration of the phosphate buffer can be 10-100 mM, preferably 20-90 mM, 30-80 mM, 40-70 mM, 40-60 mM or 45-55 mM, for example 50 mM. The polysaccharide in step 2) is first ultrafiltered using a phosphate buffer to quickly remove DOC from the sample, and then desalted and concentrated with injection water to obtain a purified refined polysaccharide with a high polysaccharide recovery rate. If the phosphate buffer is not used and the ultrafiltration and diafiltration is performed directly with injection water, the desalination will not be thorough because DOC will precipitate in a lower pH environment.

[0087] The method for purifying meningococcal group A, group C, group Y, and group W135 polysaccharides of the present invention may further include a freeze-drying step, such as freeze-drying the desalted polysaccharides. The refined polysaccharides formed by freeze-drying are in solid form, which is convenient for combining with carrier proteins during subsequent vaccine preparation and is easy to store.

[0088] After the purification method of the present invention is used to purify the polysaccharides of group A, group C, group Y, and group W135 of meningococci, the obtained refined polysaccharides can be subjected to quality analysis according to the standards of the Chinese Pharmacopoeia. The quality analysis includes but is not limited to: analyzing the content of non-polysaccharide components (such as protein, nucleic acid, phosphate, sialic acid, O-acetyl and endotoxin) in the refined polysaccharides, and determining the polysaccharide recovery rate.

[0089] Meningococcal group A, group C, group Y, group W135 polysaccharides, conjugates, compositions and applications thereof

[0090] The present invention also provides meningococcal group A, group C, group Y or group W135 polysaccharide purified by the method of the present invention.

[0091] In the present invention, the purified polysaccharide obtained has one, more or all of the following characteristics:

[0092] Protein content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2 mg / g;

[0093] Nucleic acid content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2.5 mg / g;

[0094] The phosphorus content of group A polysaccharide is >80 mg / g, preferably >82 mg / g, >83 mg / g, >84 mg / g, >85 mg / g, >86 mg / g or >87 mg / g;

[0095] The sialic acid content of group C polysaccharide is >800 mg / g, preferably >802 mg / g, >803 mg / g, >804 mg / g, >805 mg / g, >806 mg / g or >807 mg / g;

[0096] The sialic acid content of group Y polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g or >570 mg / g;

[0097] The sialic acid content of the W135 group polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g, >570 mg / g, >572 mg / g, >575 mg / g, >577 mg / g, >578 mg / g or >579 mg / g;

[0098] Group A O-acetyl content ≥2.0mmol / g, preferably ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g;

[0099] Group C O-acetyl content ≥1.5mmol / g, preferably ≥1.7mmol / g, ≥1.8mmol / g, ≥1.9mmol / g, ≥2.0mmol / g, ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g;

[0100] The O-acetyl content of group Y is ≥0.3 mmol / g, preferably ≥0.4 mmol / g, ≥0.5 mmol / g, ≥0.6 mmol / g, ≥0.8 mmol / g, ≥1.0 mmol / g, ≥1.1 mmol / g or ≥1.2 mmol / g;

[0101] W135 group O-acetyl content ≥ 0.3mmol / g;

[0102] Endotoxin is <12.5 EU / μg, preferably <12 EU / μg, <11 EU / μg, <10 EU / μg, <9 EU / μg, <8 EU / μg, <7 EU / μg or <6 EU / μg.

[0103] The present invention also provides a conjugate, wherein the conjugate comprises one or more selected from the following:

[0104] (1) Meningococcal group A polysaccharide and a carrier protein conjugated thereto,

[0105] (2) Meningococcal group C polysaccharide and carrier protein conjugated thereto,

[0106] (3) Meningococcal group Y polysaccharide and carrier protein conjugated thereto,

[0107] (4) Meningococcal W135 group polysaccharide and carrier protein conjugated thereto.

[0108] The present invention also provides a composition comprising:

[0109] (A) any one, a combination of multiple or all of meningococcal group A polysaccharide, group C polysaccharide, group Y polysaccharide, group W135 polysaccharide; and / or

[0110] (B) A conjugate of any one, more than one or all of (1) to (4).

[0111] The meningococcal group A, group C, group Y or group W135 polysaccharide of the present invention, or the composition, can be used to prepare a vaccine. For example, the meningococcal group A, group C, group Y or group W135 polysaccharide of the present invention, or the composition, can be combined with a suitable carrier to prepare a vaccine against meningococci. The meningococcal group A, group C, group Y or group W135 polysaccharide present in the vaccine can be in the form of a free polysaccharide, or as a component of a conjugate in which the polysaccharide is covalently linked to a carrier protein.

[0112] Typically, the carrier protein is any protein or fragment thereof that is acceptable to an individual and can induce an immune cell (e.g., T-cell) dependent response. The protein as a carrier typically has at least one free amino group for conjugation with a polysaccharide. In some embodiments, the carrier protein is any natural or recombinant bacterial protein, and itself is an immunogen that induces a T-cell dependent response in young and adult mammals. Exemplary carrier proteins include, but are not limited to, toxins or toxoids of tetanus toxoid, cholera toxin, diphtheria toxoid, Pseudomonas, Staphylococcus, Streptococcus, pertussis, and enterotoxigenic bacteria including Escherichia coli.

[0113] The vaccine described in the present invention can be used alone or mixed with adjuvants, other vaccines, etc. Exemplary adjuvants include but are not limited to: aluminum hydroxide, aluminum sulfate, formylmethionyl peptide, complete Freund's adjuvant, and incomplete Freund's adjuvant.

[0114] The beneficial effects of the present invention include:

[0115] The purification method of the present invention uses 0.5-2% sodium deoxycholate to pretreat crude polysaccharide, uses a Rigose MMA chromatography column and a Rigose DEAE chromatography column in series for purification, and adopts an ultrafiltration desalting method to remove the process of using a chromatography column for desalting. The synergistic effect of multiple improved steps shortens the process time, saves the process cost, and obtains refined polysaccharide that meets the pharmacopoeia standards and has a higher polysaccharide recovery rate.

[0116] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0117] Experimental equipment and materials:

[0118] 1. Main equipment:

[0119] Table 1. Main equipment

[0120] Device Name Device Model factory Electronic balance JA41002 Shanghai Sunny Hengping Magnetic stirrer MS-S Dalong Xingchuang Experimental Instruments Chromatography system AKTA Avant 150 Cytiva Ultrafiltration fixture CM018LV PALL Ultrafiltration membrane package OS100T02 PALL

[0121] 2. Main materials:

[0122] 2.1 Polysaccharide: Group A, Group C, Group W135, Group Y crude polysaccharides were prepared by the following method: Group A, Group C, Group W135, Group Y Neisseria meningitidis purchased from the China Food and Drug Inspection Institute were used, and after fermentation and culture, 10% CTAB solution (mass percentage) was slowly added to the inactivated centrifugal supernatant of the fermentation broth to make the final concentration of the supernatant 0.1% (mass percentage), and stirred while adding, and then stood for more than 3 hours. After standing, continuous flow centrifugation was performed by a tubular centrifuge, and the centrifugal speed was 15000rpm. The supernatant was centrifuged and the precipitate was collected, which was the composite polysaccharide and weighed. 4mL of 2M calcium chloride solution was added to each gram of composite polysaccharide, and stirred for 1.5 hours until completely dissolved. According to its volume, a pre-cooled 95% (volume ratio) ethanol solution was added to make the final concentration of ethanol in the solution 25%, and after mixing, it was stood at 2-8°C and precipitated for more than 3 hours. After precipitation, the mixed solution is centrifuged to remove the precipitate, and the centrifugation parameters are 7000rpm, 8°C, and 40min. 95% ethanol solution is added to the centrifuged supernatant to make its final concentration 75% (volume ratio), and it is allowed to stand at 2-8°C for 8-24 hours. After precipitation, the mixed solution is centrifuged to remove the supernatant, and the centrifugation parameters are 7000rpm, 8°C, and 15min. After centrifugation, the precipitate collected is the crude polysaccharide.

[0123] 2.2 Main reagents: 1% sodium deoxycholate (DOC) Tris-HCl buffer pH 8.0, 2% sodium deoxycholate (DOC) Tris-HCl buffer pH 8.0, 20 mM Tris-HCl buffer pH 8.0, 50 mM phosphate buffer (PB) pH 8.0, chromatography media Rigose MMA, Rigose DEAE.

[0124] Table 2. Chromatographic media and reagent information

[0125] Chromatography media and reagents Specification factory Rigose MMA PrePack GL 50 / 400 Qianchun Biology Rigose DEAE PrePack GL 50 / 200 Qianchun Biology DOC 500g / bottle Sigma Tris base 1000g / bottle Suzhou subfamily Sodium dihydrogen phosphate monohydrate 500g / bottle Hunan Jiudian Hongyang Disodium hydrogen phosphate anhydrous 500g / bottle Hunan Jiudian Hongyang

[0126] Example 1: Chromatographic purification process for crude polysaccharides of novel meningococcal groups A, C, Y and W135

[0127] In this embodiment, a chromatography purification process for crude polysaccharides of novel meningococcal group A, group C, group Y, and group W135 is provided, comprising:

[0128] 1. Sample pretreatment:

[0129] Weigh the raw sugars of group A, group C, group W135, and group Y at a concentration of 5 mg / mL and dissolve them in 20mM Tris-HCl (pH 8.0). After they are completely dissolved, add an equal volume of 2% DOC 20mM Tris-HCl (pH8.0) buffer solution and mix well to obtain raw sugar samples. The raw sugar reconstituted solutions of group A, C, Y, and W135 are fully reacted at room temperature for more than 3 hours. The samples are centrifuged at 9000 rpm and 8°C for 20 minutes, clarified and filtered with a 0.45 μm filter membrane, and then the subsequent operations are carried out. Due to the action of the anionic detergent sodium deoxycholate, polysaccharides can be separated from proteins, nucleic acids, and endotoxins, and precipitated. After centrifugation and filtration with a 0.45 μm filter membrane, the analyzed proteins, nucleic acids, and endotoxins are partially removed.

[0130] 2. Chromatographic purification:

[0131] PrePack GL 50 / 400 Rigose MMA and PrePack GL 50 / 200 Rigose DEAE were connected in series, pre-equilibrated with 20 mM Tris-HCl pH 8.0 buffer, and the chromatography columns were equilibrated with 1% DOC 20 mM Tris-HCl pH 8.0 buffer solution.

[0132] The volume (CV) of the chromatography column after series connection was calculated, and the sample was loaded at 20% CV. The crude polysaccharides of group A, group C, group W135, and group Y were loaded onto the column. Rigose MMA and Rigose DEAE chromatography media were connected in series, and the flow-through mode was adopted. The polysaccharides, proteins, and nucleic acids were monitored at the ultraviolet absorption peaks of 206, 280, and 260 nm. The polysaccharide collection peak, group A λ206 The peak starts to close at 120mAu, and after the UV peak λ 206 120mAu stops collecting peak; C, Y, W135 group λ 206 When λ 280 or 260 The peak is stopped when the value is 20mAu. The polysaccharide chromatography harvest liquid is ultrafiltered and washed with 50mM PB (PB is a phosphate buffer, the formula is as follows: accurately weigh 0.37g of monosodium dihydrogen phosphate monohydrate and 6.72g of disodium hydrogen phosphate, add an appropriate amount of injection water to fully dissolve, adjust the pH to 8.0, and add injection water to 1L, that is, first use 50mM PB pH8.0 buffer) pH8.0 solution to remove sodium deoxycholate, and then use injection water to ultrafilter and wash to remove PB. The polysaccharide harvest liquid is desalted by ultrafiltration. Most of the proteins and nucleic acids are bound to the column through the action of anions and cations, and are washed out through the high salt elution stage. After the polysaccharide ultrafiltrate is pre-frozen, it is placed in a freeze dryer for freeze drying to obtain refined polysaccharides of group A, group C, group W135, and group Y.

[0133] Example 2, Chromatographic Purification Results, Refined Polysaccharide Detection Results

[0134] According to the method of Example 1, crude polysaccharides of novel meningococcal group A, group C, group Y, and group W135 were purified by chromatography in three batches. The chromatograms are shown in FIG. Figure 1-12 shown.

[0135] After chromatography purification, the refined polysaccharides were tested for the test indicators of each group of samples according to the methods and standards of the Chinese Pharmacopoeia. According to the standards of the Chinese Pharmacopoeia, the refined polysaccharides should meet the following conditions:

[0136] Protein content <8mg / g;

[0137] Nucleic acid content <8mg / g;

[0138] Phosphorus / sialic acid content: phosphorus content of group A polysaccharide>80mg / g, sialic acid content of group C polysaccharide>800mg / g, sialic acid content of group Y polysaccharide>560mg / g, sialic acid content of group W135 polysaccharide>560mg / g;

[0139] O-acetyl content: Group A ≥ 2.0 mmol / g, Group C ≥ 1.5 mmol / g, Group Y or Group W135 ≥ 0.3 mmol / g;

[0140] Endotoxin <12.5EU / μg.

[0141] The actual test results are shown in Table 3.

[0142] Table 3. Recovery and assay results of three batches of chromatographically purified polysaccharides from group A, group C, group Y, and group W135

[0143]

[0144] Note: Not detected means below the detection limit of the instrument.

[0145] The results are shown in Table 3. Using the method of the present invention, the polysaccharide recovery rate for meningococcal group A, group C, group W135, and group Y can reach more than 80%, and the impurity contents such as protein, nucleic acid and endotoxin are all lower than the standards specified in the pharmacopoeia.

[0146] Example 3: Investigation of DOC concentration and treatment time

[0147] In this example, the treatment concentration (mass percentage) and treatment time of DOC during the pretreatment of the sample were investigated according to the method of Example 1. The results are shown in Table 4.

[0148] Table 4 Effects of DOC treatment concentration and time on polysaccharide recovery

[0149]

[0150]

[0151] The results showed that DOC pretreatment concentration of 0.5% to 1.5% and treatment time of 3 to 12 hours can achieve good separation effect. In addition, crude polysaccharides can be well separated in 0.5% to 1.5% DOC buffer system.

[0152] Example 4: Study of Chromatographic Media

[0153] In this example, according to the method of Example 1, the polysaccharide was purified by chromatography using a domestic chromatography medium or an imported chromatography medium (Table 4). Except for the following steps, the other steps were the same as those in Example 1:

[0154] Domestic chromatography medium: Rigose MMA and Rigose DEAE are connected in series. PrePack GL 50 / 400 Rigose MMA and PrePack GL 50 / 200 Rigose DEAE are connected in series, pre-equilibrated with 20mM Tris-HCl pH8.0 buffer, and the chromatography column is equilibrated with 1% DOC 20mM Tris-HCl pH8.0 buffer solution. The polysaccharide chromatography harvest liquid is ultrafiltered and washed with 50mM phosphate buffer (PB) pH8.0 solution, and then ultrafiltered and washed with injection water, and the polysaccharide harvest liquid is desalted by ultrafiltration.

[0155] Imported chromatography medium: Capto adhere and Capto DEAE were connected in series, pre-equilibrated with 20 mM Tris-HCl pH 8.0 buffer, and the chromatography column was equilibrated with 1% DOC 20 mM Tris-HCl pH 8.0 buffer solution. The harvested liquid after chromatography was desalted using Sephodex G25.

[0156] Table 5. Comparison of domestic and imported chromatography media

[0157]

[0158] The polysaccharides were purified by chromatography using domestic chromatography media or imported chromatography media, and the test results of polysaccharide recovery rate, protein content, nucleic acid content, phosphorus / sialic acid content, O-acetyl and endotoxin are shown in Table 5.

[0159] Table 6 Comparison of refined sugar detection indicators of domestic and imported chromatography media

[0160]

[0161] Note: Not detected means below the detection limit of the instrument.

[0162] The results showed that for meningococcal group A, group C, group W135, and group Y, the chromatographic medium RigoseMMA from a domestic manufacturer was connected in series with Rigose DEAE, and the polysaccharide was separated from other impurities by one-step chromatography in a flow-through mode. The harvested liquid was subjected to ultrafiltration, filtration, and freeze-dried to obtain the refined polysaccharide. Its detection index met the requirements of the pharmacopoeia, and the impurity (protein, nucleic acid) removal rate and polysaccharide recovery rate were better than those using imported chromatographic media. Moreover, since domestic chromatographic media were used, molecular exclusion desalting was not performed, and the production cost was reduced.

[0163] Example 5: Investigation of ultrafiltration desalination steps

[0164] In this example, the ultrafiltration step was explored according to the method of Example 1, and was further compared and analyzed with the literature method.

[0165] The literature method was based on the Sephodex G25 molecular exclusion chromatography desalting method described in "Establishment of the process for purifying group A, C, Y, and W135 meningococcal capsular polysaccharides by chromatography", Wu Qingsheng, Sun Fangfang, and Xiao Zhanrong. International Journal of Biological Products, Vol. 42, No. 2, April 2019, and "Removal of impurities from crude sugar of group A meningococcal polysaccharide vaccine by chromatography", Jie Hongyan, Wang Jiyuan, Liu Jing, Wang Yijun, Zhao Zhanyong, Wu Yuntao, Miao Jingyun, and Wu Yuanyuan. China Biotechnology, 2010 30(8): 88-92.

[0166] Detection method of DOC residue: accurately measure 1mL of the test solution and place it in a test tube, add 14mL of 43.5% sulfuric acid solution to the test tube, mix well, place it in a 70℃ water bath for 20 minutes, take it out, and cool it to room temperature. Accurately measure 0.0mL, 0.1mL, 0.2mL, 0.4mL, and 0.8mL of sodium deoxycholate reference solution, place them in test tubes respectively, add 60% acetic acid solution to 1mL each, add 14mL of 43.5% sulfuric acid solution to each tube, shake well, place it in a 70℃ water bath for 20 minutes, take it out, cool it to room temperature, and measure the absorbance value at a wavelength of 387nm. Perform a linear regression with the corresponding absorbance values ​​of the sodium deoxycholate reference series concentration (μg / mL) to obtain the linear regression equation. Substitute the absorbance value of the test solution into the linear regression equation to calculate the concentration of DOC in the test sample (μg / mL).

[0167] Table 7. Comparison between the method of the present invention and the method in the literature

[0168]

[0169]

[0170] Note: Not detected means below the detection limit of the instrument.

[0171] The results showed that when ultrafiltration desalination was performed, 50mM PB was used for ultrafiltration and diafiltration, which could quickly remove DOC from the sample, and then water for injection was used for desalination and concentration. If water for injection was used directly for ultrafiltration and diafiltration, DOC would precipitate in a lower pH environment, resulting in incomplete desalination. After desalination, refined polysaccharides were obtained by freeze-drying, with high purity and polysaccharide recovery rate. After testing, the polysaccharide recovery rate could reach more than 80%, and the impurity contents such as protein, nucleic acid and endotoxin were all lower than the standards specified in the pharmacopoeia.

[0172] In summary, the present invention establishes a novel chromatography purification process for ACYW135 group meningococcal polysaccharide. Compared with the literature method, the chromatography medium is replaced, a desalting chromatography step is removed, and the quality and recovery rate of the polysaccharide are improved, the product quality is effectively improved, and the production time and production cost are shortened. The process can be used for the purification of A, C, Y, and W135 group meningococcal polysaccharides.

[0173] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for purifying meningococcal polysaccharide, comprising: 1) pre-treating meningococcal polysaccharide with 0.5-2% sodium deoxycholate; 2) using a Rigose MMA chromatography column and a Rigose DEAE chromatography column in series to purify the polysaccharide pretreated in step 1); 3) using a phosphate buffer to ultrafilter and desalt the polysaccharide in step 2) to obtain purified polysaccharide; Wherein, the meningococcal polysaccharide is meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, or meningococcal group W135 polysaccharide.

2. The purification method according to claim 1, characterized in that In step 1), the content of sodium deoxycholate is 0.5-2%, preferably 0.6-1.9%, 0.7-1.8%, 0.8-1.7%, 0.9-1.6% or 1.0-1.5%, wherein the content is expressed as a mass percentage; and / or, The sodium deoxycholate treatment time is 1 to 15 hours, preferably 2 to 14 hours, 3 to 13 hours, 3 to 12 hours, 5 to 12 hours, 6 to 12 hours, 7 to 12 hours, 8 to 12 hours or 9 to 12 hours; and / or, The sodium deoxycholate is prepared in a buffer system, which further comprises 1-100 mM Tris-HCl buffer, preferably 2-90 mM, 3-80 mM, 5-50 mM, 8-40 mM, 10-30 mM or 15-25 mM; the pH value of the buffer solution is 6.0-9.0, preferably 6.5-8.5, 6.5-8.0, 7.0-8.5 or 7.5-8.

0.

3. The purification method according to claim 1 or 2, characterized in that The step 2) further comprises: balancing the chromatography column with a buffer; Preferably, the chromatography column is pre-equilibrated with a buffer solution that does not contain sodium deoxycholate, and then the chromatography column is equilibrated with a buffer solution that contains 0.5-2% sodium deoxycholate; More preferably, the buffer solution is 1-100 mM Tris-HCl buffer, more preferably 2-90 mM, 3-80 mM, 5-50 mM, 8-40 mM, 10-30 mM or 15-25 mM; the pH value of the buffer solution is 6.0-9.0, more preferably 6.5-8.5, 6.5-8.0, 7.0-8.5 or 7.5-8.

0.

4. The purification method according to any one of claims 1 to 3, characterized in that In step 3), the ultrafiltration desalination comprises: ultrafiltration of the polysaccharide in step 2) using a phosphate buffer, and then ultrafiltration elution with water for injection to remove the phosphate buffer; and / or, In step 3), the concentration of the phosphate buffer is 10-100 mM, preferably 20-90 mM, 30-80 mM, 40-70 mM, 40-60 mM or 45-55 mM; and / or, The step 3) further comprises: freeze-drying the polysaccharide solution after ultrafiltration and desalination, thereby obtaining a purified and refined polysaccharide in a solid form.

5. The purification method according to any one of claims 1 to 3, characterized in that In step 1), the crude meningococcal polysaccharide is a crude meningococcal polysaccharide obtained by separation from fermentation broth of meningitis pathogenic bacteria; Preferably, the meningitis pathogenic bacteria include or are: Neisseria meningitidis; More preferably, the meningitis pathogenic bacteria include or are selected from: Neisseria meningitidis serogroups A, C, Y, and W135; Preferably, the step of separating crude polysaccharides using fermentation broth of meningococcal pathogenic bacteria comprises: fermenting and culturing group A, group C, group Y or group W135 meningococcal pathogenic bacteria, inactivating the fermentation broth and then centrifuging it, adding a cationic detergent and a polysaccharide precipitant to the centrifugal supernatant, and separating and obtaining crude meningococcal polysaccharides; more preferably, the cationic detergent is CTAB and / or the polysaccharide precipitant is ethanol; more preferably, after adding CTAB and ethanol, the mass percentage of CTAB in the solution is 0.1±0.05%, and the volume percentage of ethanol is 75±1%.

6. A meningococcal polysaccharide obtained by the purification method according to any one of claims 1 to 5; wherein The meningococcal polysaccharide is meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, or meningococcal group W135 polysaccharide.

7. The meningococcal polysaccharide according to claim 6, characterized in that The meningococcal polysaccharide has one, more or all of the following characteristics: Protein content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2 mg / g; Nucleic acid content <8 mg / g, preferably <7 mg / g, <6 mg / g, <5 mg / g, <4 mg / g, <3 mg / g or <2.5 mg / g; The phosphorus content of group A polysaccharide is >80 mg / g, preferably >82 mg / g, >83 mg / g, >84 mg / g, >85 mg / g, >86 mg / g or >87 mg / g; The sialic acid content of group C polysaccharide is >800 mg / g, preferably >802 mg / g, >803 mg / g, >804 mg / g, >805 mg / g, >806 mg / g or >807 mg / g; The sialic acid content of group Y polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g or >570 mg / g; The sialic acid content of the W135 group polysaccharide is >560 mg / g, preferably >562 mg / g, >563 mg / g, >564 mg / g, >565 mg / g, >566 mg / g, >567 mg / g, >568 mg / g, >569 mg / g, >570 mg / g, >572 mg / g, >575 mg / g, >577 mg / g, >578 mg / g or >579 mg / g; Group A O-acetyl content ≥2.0mmol / g, preferably ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g; Group C O-acetyl content ≥1.5mmol / g, preferably ≥1.7mmol / g, ≥1.8mmol / g, ≥1.9mmol / g, ≥2.0mmol / g, ≥2.1mmol / g, ≥2.2mmol / g or ≥2.3mmol / g; The O-acetyl content of group Y is ≥0.3 mmol / g, preferably ≥0.4 mmol / g, ≥0.5 mmol / g, ≥0.6 mmol / g, ≥0.8 mmol / g, ≥1.0 mmol / g, ≥1.1 mmol / g or ≥1.2 mmol / g; W135 group O-acetyl content ≥ 0.3mmol / g; Endotoxin is <12.5 EU / μg, preferably <12 EU / μg, <11 EU / μg, <10 EU / μg, <9 EU / μg, <8 EU / μg, <7 EU / μg or <6 EU / μg.

8. A conjugate comprising one or more selected from the following: (1) Meningococcal group A polysaccharide and a carrier protein conjugated thereto, (2) Meningococcal group C polysaccharide and carrier protein conjugated thereto, (3) Meningococcal group Y polysaccharide and carrier protein conjugated thereto, (4) Meningococcal group W135 polysaccharide and carrier protein conjugated thereto; in, The meningococcal group A polysaccharide, meningococcal group C polysaccharide, meningococcal group Y polysaccharide, and meningococcal group W135 polysaccharide are meningococcal polysaccharides as claimed in claim 5 or 6, or are obtained by the purification method as claimed in any one of claims 1 to 4; Preferably, the carrier protein is a protein or a fragment thereof that is acceptable to an individual and can induce an immune cell-dependent response; more preferably, the carrier protein has at least one free amino group for conjugation with a polysaccharide; more preferably, the carrier protein comprises: tetanus toxoid, cholera toxin, diphtheria toxoid, toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis and enterotoxin-producing bacteria including Escherichia coli; more preferably, the tetanus toxoid, cholera toxin, diphtheria toxoid, toxins or toxoids of Pseudomonas, Staphylococcus, Streptococcus, pertussis and enterotoxin-producing bacteria including Escherichia coli comprise mutants thereof.

9. A composition comprising: The meningococcal polysaccharide according to claim 6 or 7, and / or the conjugate according to claim 8; Preferably, the composition further comprises an adjuvant; more preferably, the adjuvant comprises one or more selected from the group consisting of aluminum hydroxide, aluminum phosphate, monophosphoryl lipid A, QS21, CpG, stearoyl tyrosine and Freund's adjuvant.

10. Use of the meningococcal polysaccharide according to claim 6 or 7, or the conjugate according to claim 8, or the composition according to claim 9, in the preparation of a medicament for providing protection against bacterial meningitis pathogen infection; preferably, the medicament is a vaccine; Preferably, the meningitis pathogenic bacteria include or are: Neisseria meningitidis; more preferably, the meningitis pathogenic bacteria include or are selected from: Neisseria meningitidis serogroups A, C, Y, and W135.

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