Method for purifying group C meningococcus capsular polysaccharide

Abstract

The invention provides a method for purifying group C meningococcus capsular polysaccharide. The method comprises a rough preparation step and a refining step, wherein in the refining step, two-step elution purification or flow-through combined elution purification is adopted for refining. The refined capsular polysaccharide prepared by the invention meets part of quality standards required by pharmacopeia, and does not use organic solvents and toxic reagents, such as ethanol, phenol, acetone, cetyltrimethylammonium bromide (CTAB) and the like, so that the harm of the capsular polysaccharide to human bodies and the environment is avoided. The method provided by the invention also has the advantages of simple process steps, short process time consumption, reduced production cost, high safety and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for purifying polysaccharides from the capsular region of group C meningococci. Background Technology

[0002] Neisseria meningitidis ( Neisseria meningitidis , N . meningitis Neisseria meningitidis, also known as meningococcus, is a Gram-negative coccus that resides in the human upper respiratory tract, and humans are its only host. Infection can cause bacterial meningitis and sepsis, making it a pathogen with extremely high rates of disability and death.

[0003] Based on serological classification, meningococci can be divided into 13 serogroups, but five serogroups (A, B, C, Y, and W135) are the main causes of epidemic meningitis. Capsular polysaccharides are the main virulence factors of meningococci, and due to their strong antigenicity, they have been widely used in the development of meningococcal polysaccharide vaccines and polysaccharide conjugate vaccines.

[0004] Currently, the extraction of meningococcal capsular polysaccharides primarily employs methods using highly toxic organic solvents such as ethanol, phenol, acetone, CTAB, and Triton X. These methods pose significant risks of residual toxicity, environmental and human harm, complex processes, and long processing times. Triton X degradation product nonylphenol (NP) is a potent endocrine disruptor (EDC) that persists in the environment and exhibits strong bioaccumulation. Long-term exposure to NP may lead to feminization in aquatic organisms and poses potential harm to the human reproductive system. Due to the toxicity of its degradation products to aquatic organisms, it has been banned in Europe. Meanwhile, next-generation bioprocesses are currently undergoing studies challenging the ban on Triton X-100. Furthermore, capsular polysaccharides extracted with organic solvents such as phenol suffer from molecular weight inhomogeneity and significant batch-to-batch variability; their purity and structural integrity directly affect vaccine efficacy.

[0005] Existing technologies, such as patents CN106191164A and CN106117388A, disclose a method for high-density saccharification of group C meningococci. The fermentation broth supernatant requires steps including CTAB precipitation, calcium chloride dissociation, ethanol precipitation, cold phenol extraction with sodium acetate, and membrane ultrafiltration to obtain purified polysaccharides. The endotoxin content of the capsular polysaccharides obtained by this method is <6.25 EU / μg, and the polysaccharide yield is between 20.7% and 24.3%.

[0006] Another patent, CN115478033A, discloses a method for fermenting and culturing meningococci, along with its fermentation broth, capsular polysaccharide, and applications. The fermentation broth, after centrifugation to collect the supernatant, undergoes a complex process involving CTAB precipitation, calcium chloride dissociation, ethanol precipitation, filtration clarification, further ethanol precipitation, cold phenol extraction with sodium acetate, ultrafiltration, and a third ethanol precipitation to obtain purified polysaccharide. This process yields a final capsular polysaccharide yield of 35–166 mg / L and an endotoxin content of 8–12.5 EU / μg.

[0007] Patent CN106146679B discloses a method for purifying capsular polysaccharides using Tritonx 114, although it does not use phenol. However, this patent still uses organic solvents such as anhydrous ethanol and acetone, as well as toxic reagents, which still pose foreseeable risks and hazards to human health and the environment.

[0008] Therefore, there is an urgent need in this field for a more advanced and safer polysaccharide extraction technology from meningococci to develop more reliable, safe, and effective vaccines, thereby more effectively preventing and controlling the occurrence of epidemic meningitis. Summary of the Invention

[0009] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and provide a method for preparing polysaccharide from group C meningococcus capsularis. This method is simple, eliminates organic solvents and toxic reagents, is convenient to prepare, has low cost, and is suitable for large-scale industrial production.

[0010] To address the aforementioned technical problems, this invention provides a method for purifying capsular polysaccharides from group C meningococci. The method comprises two steps: crude purification and purification. Purification is performed using a two-step elution purification process or a flow-through combined elution purification process. The method satisfies one or more of the following:

[0011] 1) Use a continuous flow centrifuge or an angle rotor centrifuge for coarse processing;

[0012] 2) The buffer solution used for purification contains Tris-HCl and NaCl; and,

[0013] 3) Purification was performed using a multi-mode strong anion exchange chromatography column.

[0014] In some embodiments, the crude step of the method involves using a depth filter, membrane pack, or hollow fiber filter, or centrifugation to collect the clarified permeate or centrifugal supernatant.

[0015] In some implementations, the pore size of the depth filter is 1.5 to 3.0 μm.

[0016] In some implementations, the membrane pore size is 500 kDa.

[0017] In some embodiments, the hollow fiber has a pore size of 0.65 μm or 0.2 μm.

[0018] In some implementations, the centrifugation method is continuous flow centrifugation or angular rotor centrifugation.

[0019] In some embodiments, the crude step of the method involves centrifugation at 10,000-16,000 rpm using a continuous flow centrifuge.

[0020] In some implementations, a continuous flow centrifuge is used to centrifuge 2-4 times.

[0021] In some implementations, a continuous flow centrifuge at 12,000-16,000 rpm is used for centrifugation.

[0022] In some implementations, a continuous flow centrifuge is used to centrifuge at 14,000 rpm, and / or, centrifuge three times.

[0023] In some embodiments, the crude step of the method involves centrifugation at 9000-13000 rpm using an angle rotor centrifuge.

[0024] In some implementations, centrifugation is performed using an angle rotor centrifuge for 0.5-1.5 hours.

[0025] In some implementations, an angle rotor centrifuge is used to centrifuge at 10,000-12,000 rpm.

[0026] In some implementations, centrifugation is performed using an angle rotor centrifuge at 11,000 rpm and / or for 1 hour.

[0027] In some implementations, centrifugation is performed once using an angle rotor centrifuge.

[0028] In some implementations, the “two-step elution purification” uses a multi-mode strong anion exchange chromatography column for purification.

[0029] In some implementations, the "two-step elution purification" includes a first-step purification and a second-step purification, which respectively include sample conditioning, sample loading, rinsing, elution, buffer system replacement, and filtration.

[0030] In some implementations, a column equilibration step is also included before sample loading, wherein the equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl.

[0031] In some implementations, the buffer used for sample conditioning contains 0.05-0.15 mol / L Tris-HCl and 1.5-1.8 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 1.65 mol / L NaCl.

[0032] In some embodiments, the rinsing buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl.

[0033] In some embodiments, the elution buffer contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl.

[0034] In some implementations, the buffer system is replaced by membrane packing for concentration, preferably using 3-30 kDa membrane packs.

[0035] In some implementations, the buffer system is replaced by concentration using an 8-12 kDa membrane pack.

[0036] In some implementations, the buffer system is first replaced with NaCl solution and then with pure water, wherein the NaCl solution is preferably a 0.7%-1.0% NaCl solution; the volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9).

[0037] In some implementations, membrane filtration is used.

[0038] In some implementations, filter membranes with a diameter of less than 0.3 μm are used.

[0039] In some implementations, filtration is performed using a 0.22 μm or 0.23 μm filter membrane.

[0040] In some implementations, during the sample conditioning step, the volume ratio of the crude sample to the buffer solution is (3-5):1, for example, 4:1.

[0041] In some implementations, during equilibration, 2-3 CV is equilibrated using a buffer solution, preferably 3 CV.

[0042] In some implementations, during rinsing, buffer solution is used to rinse 2-3 CV, preferably 2 CV.

[0043] In some implementations, elution is performed using a buffer solution to elute 2-3 CV, preferably 3 CV.

[0044] In some implementations, 10KD membrane packs are used in the buffer system replacement.

[0045] In some implementations, during buffer system replacement, NaCl solution is used for 1-3 replacements, preferably 2, and pure water is used for 3-5 replacements, preferably 4.

[0046] In some implementations, filtration is performed using a filter membrane with a diameter of less than 0.23 μm.

[0047] In some implementations, the "flow-through combined elution purification" includes a first-step purification and a second-step purification. The first-step purification includes sample conditioning, sample loading, flow-through collection, buffer system replacement, and filtration. The second-step purification includes sample conditioning, sample loading, rinsing, elution, buffer system replacement, and filtration.

[0048] The "flow-through combined elution purification" method uses a multi-mode strong anion exchange chromatography column for purification.

[0049] In some embodiments, the multimode strong anion exchange chromatography column includes Diamond MIX-AMustang, Capto Adhere, Toyopearl MX-Trp-650M, POROS XQ, and MEP HyperCel chromatography columns.

[0050] In some implementations, the first step of purification is purification using a chromatography column.

[0051] In some implementations, the first step uses column chromatography columns including Diamond Mix-A Mustang, CaptoDEAE (HiScreen), SOURCE 30Q, hydroxyapatite 80B, Monomix MC60, Phenyl 6FF highSub-HS, and Ni Sepharose Excel columns.

[0052] In some implementations, the chromatography column is Diamond Mix-A Mustang.

[0053] In some implementations, a column equilibration step is included before sample loading, wherein the equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, containing 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl.

[0054] In some implementations, the buffer solution used in sample conditioning contains 0.05-0.15 mol / L Tris-HCl and 2.5-3.0 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 2.75 mol / L NaCl.

[0055] In some implementations, the buffer solution used in flow-through collection contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl.

[0056] In some implementations, membrane packs are used for concentration during buffer system replacement, preferably 3-30 kDa membrane packs.

[0057] In some implementations, during buffer system replacement, NaCl solution is used first for replacement, followed by pure water replacement. The NaCl solution is preferably a 0.7%-1.0% NaCl solution. The volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9).

[0058] In some implementations, filtration is performed using a filter membrane, preferably a membrane with a diameter of less than 0.3 μm.

[0059] In some implementations, the first purification step satisfies one or more of the following:

[0060] 1) During sample preparation, the volume ratio of crude sample to buffer solution is (3-5):1, for example, 4:1;

[0061] 2) During equilibration, use buffer solution to equilibrate at 2-3 CV, preferably 3 CV;

[0062] 3) During flow-through collection, rinse with buffer for 2-3 cycles, preferably 2 cycles;

[0063] 4) In the replacement of the buffer system, a 10KD membrane is used;

[0064] 5) In the buffer system replacement, NaCl solution is used for 1-3 replacements, preferably 2; pure water is used for 3-5 replacements, preferably 4; and,

[0065] 6) Use a filter membrane with a diameter of less than 0.23 μm for filtration.

[0066] In some implementations, the first step of purification uses a flow-through mode, wherein the sample conditioning buffer is 0.1 mol / L Tris-HCl (pH 8.0) containing 2.75 mol / L NaCl buffer, and the volume ratio of sample to conditioning buffer is 4:1; after sample loading, the sample is washed for 2 CV with 0.02 mol / L Tris-HCl (pH 8.0) containing 0.55 mol / L NaCl buffer, and the flow-through is collected.

[0067] In some embodiments, the first step of purification uses an elution mode, wherein the sample conditioning buffer for the elution mode is 0.1 mol / L Tris-HCl (pH 8.0) containing 1.65 mol / L NaCl buffer, with a sample to conditioning buffer volume ratio of 4:1. After sample conditioning, the sample is loaded; after loading, it is washed for 2 CV with 0.02 mol / L Tris-HCl (pH 8.0) containing 0.33 mol / L NaCl buffer; then it is eluted for 3 CV with 0.02 mol / L Tris-HCl (pH 8.0) containing 0.55 mol / L NaCl buffer, and the eluent is collected.

[0068] In some implementations, the second-step column chromatography column comprises Diamond Mix-A Mustang, Capto DEAE (HiScreen), SOURCE 30Q, hydroxyapatite 80B, Monomix MC60, Phenyl 6FF highSub-HS, and NiSepharose Excel columns.

[0069] In some embodiments, the chromatography medium is Diamond Mix-A Mustang.

[0070] In some implementations, the second purification step satisfies one or more of the following:

[0071] 1) During sample preparation, the buffer solution used contains 0.05-0.15 mol / L Tris-HCl and 1.5-1.8 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 1.65 mol / L NaCl.

[0072] 2) Before loading the sample, a column equilibration step is also included. The equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl.

[0073] 3) During rinsing, the buffer solution used contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl.

[0074] 4) During elution, the buffer solution used contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl.

[0075] 5) In the replacement of the buffer system, membrane concentration is used for concentration and replacement, preferably using 3-30KD membrane concentration;

[0076] 6) In the buffer system replacement, NaCl solution is used first, followed by pure water. The NaCl solution is preferably a 0.7%-1.0% NaCl solution. The volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9).

[0077] 7) In filtration, a filter membrane is used, preferably a filter membrane with a diameter of less than 0.3 μm.

[0078] In some implementations, the second purification step satisfies one or more of the following:

[0079] 1) During sample preparation, the volume ratio of the sample to be purified to the buffer solution is (3-5):1, for example, 4:1;

[0080] 2) During equilibration, use buffer solution to equilibrate at 2-3 CV, preferably 3 CV;

[0081] 3) Use buffer solution to rinse for 2-3 cycles, preferably 2 cycles;

[0082] 4) Elution with buffer solution for 2-3 CV, preferably 3 CV;

[0083] 5) The buffer system was concentrated and replaced using a 10KD membrane pack.

[0084] 6) In the buffer system, use NaCl solution for 1-3 displacements, preferably 2 displacements; use pure water for 3-5 displacements, preferably 4 displacements; and,

[0085] 7) Use a filter membrane with a diameter of less than 0.23 μm for filtration.

[0086] In some implementations, the sample conditioning buffer for the second step of the purification elution mode is a 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 1.65 mol / L NaCl, with a sample to conditioning buffer volume ratio of 4:1. After sample conditioning, the sample is loaded. After loading, the sample is washed for 2 CVs with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl. Then, it is eluted for 3 CVs with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl, and the eluent is collected.

[0087] In some implementations, the crude step also includes a step of collecting the supernatant after centrifugation in a continuous flow centrifuge or an angle rotor centrifuge and then concentrating and replacing it.

[0088] In some implementations, the method satisfies one or more of the following:

[0089] 1) Use hollow fiber equipment for concentration, preferably using 80-120KD hollow fiber equipment, such as using 100KD hollow fiber equipment;

[0090] 2) Use pure water for displacement, with a volume ratio of the solution to be replaced to the replacement solution of (1-2):(8-9), for example, 1:9; and,

[0091] 3) Replace the water 3-5 times, preferably 4 times;

[0092] In some implementations, concentration and displacement are performed: the collected clarified permeate or supernatant is concentrated to 1 / 5 of the fermentation broth volume using a hollow fiber apparatus, and then displacement is performed, with the concentrate finally collected.

[0093] In some embodiments, the hollow fiber has a pore size of 100 kDa.

[0094] In some implementations, the replacement buffer is purified water.

[0095] In some implementations, the replacement is a 10-fold volume replacement for desalination.

[0096] In some implementations, the number of permutations is four.

[0097] In some embodiments, the concentration and replacement process further includes a filtration step, preferably using a filter membrane with a diameter of less than 0.3 μm, such as a filter membrane with a diameter of less than 0.23 μm.

[0098] In some implementations, a 0.22 μm or 0.23 μm filter membrane is used.

[0099] In some implementations, filtration is performed: the concentrated collected liquid is filtered using a capsule filter to obtain a crude sugar solution.

[0100] In some embodiments, the pore size of the capsule filter is 0.2 μm.

[0101] In some embodiments, the crude process is carried out using fermentation broth and further includes the steps of cell lysis and release of capsular polysaccharides;

[0102] Wherein, the roughing process satisfies one or more of the following:

[0103] 1) Cells are lysed and capsular polysaccharides are released by adding DOC;

[0104] 2) Before cell lysis, the fermentation broth also includes sterilization and dilution steps, such as sterilization with formaldehyde or dilution with sodium chloride solution; and,

[0105] 3) After the release of capsular polysaccharides, a pH adjustment step is also included, for example, using acetic acid for pH adjustment.

[0106] In some implementations, DOC treatment involves adjusting the pH of the fermentation broth to 7.20 ± 0.1, adding a DOC solution, stirring, and maintaining the pH.

[0107] In some embodiments, the concentration of the DOC solution is 4.5% (w / v).

[0108] In some implementations, the final concentration of the DOC solution after addition to the fermentation broth is 0.1-0.3% (w / v), for example, 0.15% (w / v).

[0109] In some embodiments, the stirring speed is 900-1500 rpm.

[0110] In some implementations, the stirring method is mechanical stirring.

[0111] In some implementations, the stirring time is 1 hour.

[0112] In some implementations, the pH is maintained at 7.30 ± 0.10.

[0113] In some implementation schemes, the fermentation broth is sterilized: after fermentation is completed, formaldehyde is added to the fermenter for sterilization, and the pH is maintained.

[0114] In some embodiments, the final concentration of formaldehyde added is 0.5% to 1.0% (v / v).

[0115] In some implementation schemes, the final formaldehyde concentration is 0.5% (v / v).

[0116] In some implementations, the sterilization time of the formaldehyde is more than 12 hours.

[0117] In some implementations, the fermenter is maintained at a pH of 7.20 ± 0.1.

[0118] In some implementation schemes, the fermentation broth is diluted: after sterilization, the fermentation broth is collected and diluted with a high-concentration sodium chloride solution.

[0119] In some embodiments, the final concentration of the sodium chloride solution is 2 mol / L.

[0120] In some embodiments, the final concentration of the sodium chloride solution is 0.85% (w / v).

[0121] In some implementations, the fermentation broth is diluted by a factor of 2.

[0122] In some implementations, a pH adjustment step is also included after the release of the capsular polysaccharide.

[0123] In some implementations, pH is adjusted by using acetic acid to adjust the pH of the fermentation broth after DOC treatment, followed by stirring and sedimentation.

[0124] In some implementations, the pH value is 4.00 ± 0.05.

[0125] In some embodiments, the stirring speed is 900-1500 rpm.

[0126] In some implementations, the stirring method is mechanical stirring.

[0127] In some implementations, the stirring time is 1 hour.

[0128] In some implementations, the coarsening process in the method includes the following steps:

[0129] i) Cell lysis and release of capsular polysaccharides;

[0130] ii) Centrifugation and filtration; and,

[0131] iii) Concentration, replacement and filtration.

[0132] In some embodiments, the method further includes a step of refining followed by freeze-drying.

[0133] The present invention provides a method for fermenting and culturing group C meningococci, wherein the culture medium used in the method comprises soybean peptone or yeast powder, glucose, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ammonium sulfate, monosodium glutamate and coenzyme I.

[0134] In some implementations, seed culture is used to begin cultivation.

[0135] In some implementation schemes, seed culture involves opening one lyophilized working culture, reconstituted it with sterile physiological saline, and inoculating it evenly into four 2L Erlenmeyer flasks containing 1L of culture medium each, and incubating overnight at 35°C and 140rpm.

[0136] In some implementations, seed culture involves reconstituted group C meningococcal working seeds with sterile physiological saline, mixing thoroughly, and transferring the mixture into a seed culture medium. The culture is then incubated at 35°C and 140 rpm on a shaker. 600nm Stop culturing when the pH reaches approximately 0.5-2.0 to obtain the first-generation culture solution.

[0137] In some embodiments, the volume of the seed culture medium is 4L, with 1L of medium per bottle, for a total of 4 bottles.

[0138] In some embodiments, the seed culture medium comprises soybean peptone or yeast powder, glucose, sodium chloride and coenzyme I. Preferably, the concentration of soybean peptone / yeast powder is 30 g / L, the concentration of glucose is 10 g / L, and the concentration of coenzyme I is 0.02 g / L.

[0139] In some implementation schemes, fermentation culture (30L fermenter): First-generation seed culture that meets the seed culture requirements is inoculated into the 30L fermenter sequentially with the seed culture, inorganic salt solution, and coenzyme I solution under aseptic conditions (flame protection) once the fermenter temperature reaches the set temperature of approximately 35°C. The inoculation ratio is 1:3.5 (seed culture volume / fermentation basal medium volume). Fermentation process parameters are set as follows: rotation speed 100~600 rpm, aeration: depth 0.5~2 VVM; tank pressure 0.05±0.005 MPa, culture pH 7.2±0.1, culture temperature 35±1°C.

[0140] In some implementations, fermentation culture involves inoculating the obtained first-generation culture broth into a 30L fermenter containing second-generation culture medium, setting the parameters as follows: 35℃, pH 7.2, 100~600rpm, and submersible aeration 0.5~2.0VVM, and then conducting fermentation culture to cultivate A... 600nm When the value is above 1.0, start feeding. Stop culturing when the logarithmic growth phase or the early stable growth phase is reached, and collect the fermentation broth.

[0141] In some embodiments, the fermentation medium comprises soybean peptone or yeast powder, glucose, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ammonium sulfate, monosodium glutamate, and coenzyme I.

[0142] In some embodiments, the concentration of soybean peptone or yeast powder is 30 g / L, the concentration of sodium chloride is 10 g / L, the concentration of glucose is 6 g / L, the concentrations of monosodium glutamate and dipotassium hydrogen phosphate are both 5 g / L, the concentration of magnesium sulfate heptahydrate is 1.02 g / L, and the concentration of coenzyme I is 0.02 g / L.

[0143] In some embodiments, the fermentation medium is used by mixing soybean peptone or yeast powder, glucose, sodium chloride and monosodium glutamate and then sterilizing it in a fermenter. Dipotassium hydrogen phosphate and magnesium sulfate heptahydrate are prepared separately, sterilized in reagent bottles, and then added. The coenzyme is added after being sterilized by filtration through a disposable 0.2 μm sterile filter.

[0144] In some implementation schemes, feeding is carried out according to the residual sugar content (sugar supplementation begins when the residual sugar content is <4-5g / L), in batches, once per hour, in stages, and the feeding is completed during the logarithmic phase.

[0145] In some embodiments, the feed comprises a feed medium, an alkali solution, and a glucose solution.

[0146] In some embodiments, the feed culture medium comprises 30 g / L soybean peptone or yeast extract, 0.02 g / L coenzyme I, 50 mL / L amino acid mixture, 2 mL / L vitamin mixture, and 4 mL / L LT solution.

[0147] In some embodiments, the alkaline solution is a 500 g / L NaOH solution.

[0148] In some embodiments, the glucose solution is 500 g / L glucose.

[0149] In some implementations, the feeding method involves feeding once per hour.

[0150] In some embodiments, the replenishment is based on the residual sugar content. Preferably, the residual sugar content is a glucose concentration of less than 4-5 g / L.

[0151] In some implementation schemes, fermentation is stopped: A is carried out every hour. 600nm Measurements were taken, and the bacteria were stained and examined under a microscope. When the bacterial growth curve reached the late logarithmic growth phase or the early stationary phase, fermentation was stopped, and the fermentation broth was discharged and harvested.

[0152] In some implementations, the A corresponding to the cessation of fermentation 600nm The value is approximately 20-30.

[0153] Another aspect of the present invention provides a method for preparing group C meningococcal capsular polysaccharide, the method comprising purifying group C meningococcal capsular polysaccharide using the method described above provided by the present invention.

[0154] In some embodiments, the preparation method further includes fermenting and culturing group C meningococci using the method for fermenting and culturing group C meningococci provided above.

[0155] In some embodiments, the preparation method further includes fermentation culture of group C meningococci ( Neisseria meningitis The steps are as follows.

[0156] In some implementations, seed culture is used to begin cultivation, and / or, the cultivation process also includes a step of adding feed.

[0157] In some embodiments, the feed comprises a feed medium, an alkali solution, and a glucose solution.

[0158] In some embodiments, the culture medium for culturing group C meningococci contains soybean peptone or yeast extract, glucose, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ammonium sulfate, monosodium glutamate, and coenzyme I; and / or, the feed medium contains soybean peptone or yeast extract, coenzyme I, an amino acid mixture, a vitamin mixture, and T solution.

[0159] In some embodiments, the amino acid mixture comprises UDP, arginine, cysteine, and glutamine.

[0160] In some embodiments, the vitamin mixture contains biotin, folic acid, vitamin B6, vitamin B1, riboflavin, nicotinic acid, niacin, calcium pantothenate, and vitamin B12.

[0161] In some embodiments, the T solution contains MgSO4·7H2O, MnSO4·H2O, FeSO4·7H2O, CoCl·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3, NaMoO4·2H2O, and NiSO4·6H2O.

[0162] To address the aforementioned technical problems, another aspect of the present invention provides a column chromatography method for serogroup C meningococci, comprising the following steps:

[0163] i) First step: column chromatography;

[0164] ii) Second step: column chromatography; and,

[0165] iii) The third step is concentration and filtration.

[0166] In some implementations, a sample conditioning solution is added to the crude sugar solution in a certain proportion for the first step of column chromatography.

[0167] In some embodiments, the first-step column chromatography medium is Diamond Mix-A Mustang, or CaptoDEAE (HiScreen), or SOURCE 30Q, or hydroxyapatite 80B, or Monomix MC60, or Phenyl 6FFhighSub-HS, or Ni Sepharose Excel; more preferably, the chromatography medium is Diamond Mix-AMustang.

[0168] Preferably, the chromatography mode is either flow-through mode or elution mode.

[0169] In some embodiments, the sample conditioning buffer for the flow-through mode is a 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 2.75 mol / L NaCl, with a sample to conditioning buffer volume ratio of 4:1; after sample loading, the sample is rinsed 2 CV with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl, and the flow-through solution is collected.

[0170] In some embodiments, the sample conditioning buffer for the elution mode is a 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 1.65 mol / L NaCl, with a sample to conditioning buffer volume ratio of 4:1. After conditioning, the sample is loaded; after loading, it is washed for 2 CV with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl; then it is eluted for 3 CV with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl, and the eluent is collected.

[0171] In some implementations, the flow-through or eluent obtained from the first step of column chromatography is concentrated and replaced, and the concentrate is collected and filtered after completion.

[0172] In some embodiments, the membrane envelope of the concentration replacement has a pore size of 10 kDa.

[0173] In some embodiments, the replacement method involves first replacing twice with 0.85% NaCl, then replacing four times with purified water. More preferably, the replacement volume ratio of the buffer solution to the sample is 9:1.

[0174] In some implementations, the filtration method is 0.2μm capsule filter filtration.

[0175] In some implementation schemes, the filtered sample is added to a sample conditioning solution in a certain proportion for a second column chromatography step.

[0176] In some implementations, the column chromatography medium for the second step is Diamond Mix-A Mustang.

[0177] In some implementations, the chromatography mode is used as the elution mode.

[0178] In some embodiments, the sample conditioning buffer for the elution mode is a 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 1.65 mol / L NaCl, with a sample to conditioning buffer volume ratio of 4:1. After conditioning, the sample is loaded; after loading, it is washed for 2 CV with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl; then it is eluted for 3 CV with a 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl, and the eluent is collected.

[0179] In some implementations, the eluent obtained from the second-step column chromatography is concentrated and replaced, and the concentrate is collected and filtered after completion.

[0180] In some embodiments, the membrane envelope of the concentration replacement has a pore size of 10 kDa.

[0181] In some embodiments, the replacement method involves first replacing twice with 0.85% NaCl, then replacing four times with purified water. More preferably, the replacement volume ratio of the buffer solution to the sample is 9:1.

[0182] In some implementations, the filtration method is 0.2μm capsule filter filtration.

[0183] In some implementations, a freeze-drying step is also included.

[0184] In some implementations, freeze-drying involves lyophilizing the filtered refined sugar solution to obtain refined capsular polysaccharide.

[0185] In some implementations, group C meningococcal capsular polysaccharides are isolated and purified using fermentation broth produced by bacteria.

[0186] In some implementations, the centrifuged supernatant is concentrated to 1 / 5 of the fermentation broth volume using 100KD hollow fiber, and then replaced with purified water at a volume ratio of 1:4, repeated 4 times, and the concentrated replacement retentate is collected.

[0187] In some implementations, the concentrated replacement collection solution is filtered using a 0.2 μm capsule filter to collect the crude sugar solution.

[0188] In some implementations, the crude sugar solution is adjusted with a 0.1 mol / L Tris-HCl buffer containing 1.65 mol / L NaCl, with a sample to adjustment buffer volume ratio of 4:1.

[0189] In some implementations, after conditioning, the sample is loaded onto a Diamond MIX-A Mustang column, eluted for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, and then eluted for 3 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The eluent is collected at the peak.

[0190] In some implementations, the eluent is concentrated and replaced using a 10KD membrane pack.

[0191] In some implementation schemes, the solution is first replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, and then replaced four times with purified water at a volume ratio of 1:9. After the replacement is completed, the solution is filtered through a 0.2μm capsule filter.

[0192] In some implementations, the filtered sample is conditioned with a 0.1 mol / L Tris-HCl buffer containing 1.65 mol / L NaCl, with a sample to conditioning buffer volume ratio of 4:1.

[0193] In some implementations, after conditioning, the sample is loaded onto a Diamond MIX-A Mustang column, eluted for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, and then eluted for 3 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The eluent is collected at the peak.

[0194] In some implementations, the eluent is concentrated and replaced using a 10KD membrane, first by replacing it twice with 0.85% NaCl solution at a volume ratio of 1:9, and then by replacing it four times with purified water at a volume ratio of 1:9.

[0195] In some implementations, filtration is performed using a 0.2 μm capsule filter after the replacement is complete.

[0196] In some implementations, the filtered refined sugar solution is freeze-dried to obtain refined polysaccharides.

[0197] The reagents and raw materials used in this invention are all commercially available.

[0198] The positive and progressive effects of this invention include: addressing the limitations of existing methods, this invention provides a novel method for preparing meningococcal capsular polysaccharides. This method includes: diluting the fermentation broth supernatant with a salt solution, performing DOC (sodium deoxycholate) precipitation, centrifuging to collect the supernatant, concentration and displacement, and two-step column chromatography purification. The key innovation lies in the complete elimination of organic solvents and toxic reagents in the entire process, its simplicity, and reproducible yield. The final purified capsular polysaccharide has significant advantages, with its endotoxin content significantly reduced to 2 EU / ug (as low as 0.02 EU / μg), polysaccharide yield reaching 28.2%, and production reaching 78.3 mg / L.

[0199] This invention provides a method for preparing polysaccharides by removing organic solvents and toxic reagents. The method removes organic solvents, including ethanol, phenol, acetone, CTAB, Triton X, and other organic solvents and toxic reagents, thereby avoiding their harm to human health and the environment.

[0200] This invention provides a method for preparing polysaccharides with extremely low endotoxin content, which comprehensively improves the quality and safety of meningococcal vaccines.

[0201] In addition, the present invention has the advantages of simple process steps, short process time, reduced production costs and easy scale-up. Attached Figure Description

[0202] Figure 1 This is the nucleic acid electrophoresis pattern of the flow-through sample collected by chromatography in Example 7 of the present invention.

[0203] (Note: Lane 1 is the crude sugar sample, lane 2 is the Diamond Mix-A Mustang flow-through buffer, lane 3 is the CaptoDEAE flow-through buffer, lane 4 is the Source Q 30 flow-through buffer, lane 5 is the hydroxyapatite flow-through buffer, lane 6 is the Monomix MC60 Butyl flow-through buffer, lane 7 is the Phenyl 6FF highsub flow-through buffer, and lane 8 is the Ni Sepharose excel flow-through buffer.)

[0204] Figure 2 This is the SDS-PAGE electrophoresis pattern of the flow-through collected by chromatography in Example 7 of the present invention.

[0205] (Note: Lane 1 is the crude sugar sample, Lane 2 is the Diamond Mix-A Mustang flow-through buffer, Lane 3 is the CaptoDEAE flow-through buffer, Lane 4 is the Source Q 30 flow-through buffer, Lane 5 is the hydroxyapatite flow-through buffer, Lane 6 is the Monomix MC60 Butyl flow-through buffer, Lane 7 is the Phenyl 6FF highsub flow-through buffer, Lane 8 is the Ni Sepharose excel flow-through buffer, and Lane 9 is the marker.) Detailed Implementation

[0206] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0207] The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0208] Materials and sources

[0209] The group C meningococcus used in the embodiments of this invention ( Neisseria meningitidis The strain was purchased from the China Medical Microbiology Culture Collection Center (CMCC29205).

[0210] Example 1: Fermentation culture of meningococci

[0211] (1) First generation seed culture

[0212] Open one vial of lyophilized working culture, reconstitute it with 1 mL of sterile physiological saline, and inoculate 250 μL of the reconstituted culture into four 2L Erlenmeyer flasks containing 1L of culture medium each. Incubate overnight at 35°C and 140 rpm. Seed culture A 600nm The value is between 0.5 and 2.

[0213] Table 1. Formulation of First Generation Seed Culture Medium

[0214]

[0215] (2) Fermentation culture (30L fermenter)

[0216] Once the first-generation seed culture, meeting the requirements, reaches the set temperature of approximately 35°C in the fermenter, the seed culture, inorganic salt solution, and coenzyme I solution are sequentially inoculated into a 30L fermenter under aseptic conditions (flame protection). The inoculation ratio is 1:3.5 (seed culture volume / fermentation medium volume). The fermentation parameters are set as follows: rotation speed 100~600 rpm, aeration: depth 0.5~2 VVM; tank pressure 0.05±0.005 MPa, culture pH 7.2±0.1, and culture temperature 35±1°C. The fermentation medium formulation is shown in Table 2.

[0217] Feeding and sugar replenishment should be carried out according to the residual sugar content (sugar replenishment should begin when the residual sugar content is <4-5g / L). Feeding should be carried out in batches, with feeding medium (formula shown in Table 3) added once per hour. Feeding should be carried out in stages and completed during the logarithmic phase.

[0218] OD every hour 600 Measurement, staining and microscopic examination.

[0219] When the bacterial growth curve reaches the late logarithmic growth phase or the early stationary phase, or when the pH rises for 1-2 hours, fermentation is stopped, and the fermentation broth is discharged and harvested.

[0220] Table 2 Fermentation basal culture medium

[0221]

[0222] Table 3 Feeding medium formulation

[0223]

[0224] Table 4 Vitamin Mixture Formula

[0225]

[0226] Table 5 T solution

[0227]

[0228] Table 6 Amino Acid Mixture Formulation

[0229]

[0230] Table 7 Key Reagent Information

[0231]

[0232] Example 2 Extraction of crude capsular polysaccharides

[0233] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. Clarification was performed using a 1.5–3.0 μm Cobetter filter, and the clarified permeate was collected. The clarified permeate was concentrated to 1 / 5 of the fermentation broth volume using a 100 KD hollow fiber filter, and then replaced with purified water at a volume ratio of 1:9, four times. The concentrated replacement retentate was collected to obtain the concentrated replacement collected solution. The concentrated replacement collected solution was filtered through a 0.2 μm Cobetter filter to collect the crude sugar solution.

[0234] Example 3 Extraction of crude capsular polysaccharides

[0235] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. Clarification was performed using a 0.65 μm hollow fiber (WaterSep), and the clarified permeate was collected. The clarified permeate was concentrated to 1 / 5 of the fermentation broth volume using a 100 KD hollow fiber, and then replaced with purified water at a volume ratio of 1:9, four times. The concentrated replacement retentate was collected to obtain the concentrated replacement collected solution. The concentrated replacement collected solution was filtered through a 0.2 μm capsule filter (Cobetter) to collect the crude sugar solution.

[0236] Example 4 Extraction of crude capsular polysaccharides

[0237] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a final concentration of 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. Clarification was performed using a 0.2 μm hollow fiber (WaterSep), and the clarified permeate was collected. The clarified permeate was concentrated to 1 / 5 of the fermentation broth volume using a 100 KD hollow fiber, and then replaced with purified water at a volume ratio of 1:9, four times. The concentrated replacement retentate was collected to obtain the concentrated replacement collected solution. The concentrated replacement collected solution was filtered through a 0.2 μm capsule filter (Cobetter) to collect the crude sugar solution.

[0238] Example 5: Extraction of crude capsular polysaccharides

[0239] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. Clarification was performed using a 500 KD membrane (Cobetter), and the clarified permeate was collected. The clarified permeate was concentrated to 1 / 5 of the fermentation broth volume using a 100 KD hollow fiber filter, and then replaced with purified water at a volume ratio of 1:9, four times. The concentrated replacement retentate was collected to obtain the concentrated replacement collected solution. The concentrated replacement collected solution was filtered through a 0.2 μm capsule filter (Cobetter) to collect the crude sugar solution.

[0240] Example 6 Extraction of crude capsular polysaccharides

[0241] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. The mixture was centrifuged three times at 14000 rpm using a continuous flow centrifuge, and the supernatant was collected. The supernatant was concentrated to 1 / 5 of the fermentation broth volume using a 100 kDa hollow fiber filter, and then replaced with purified water at a volume ratio of 1:9, four times. The concentrated replacement retentate was collected to obtain the concentrated replacement collected solution. The concentrated replacement collected solution was filtered through a 0.2 μm Cobetter filter to collect the crude sugar solution.

[0242] The yields of MenC crude capsular polysaccharides, nucleic acids, proteins, and sugars prepared in Examples 2-6 above are shown in Table 8, indicating that the crude capsular polysaccharides prepared by centrifugation have a higher yield and a lower nucleic acid content.

[0243] Table 8. Mass of MenC polysaccharides under different processing conditions

[0244]

[0245] Example 7: Chromatography Media Screening Study

[0246] Seven media were selected: Diamond Mix-A Mustang, Capto DEAE (HiScreen), SOURCE 30Q, hydroxyapatite 80B, Monomix MC60, Phenyl 6FF highSub-HS, and Ni Sepharose Excel. The crude MenC capsular polysaccharide was dissolved to 2 mg / mL and loaded onto the sample according to the protocol in Step 9. Flow-through mode was used in all cases, and the flow-through was collected. The levels of nucleic acids, proteins, endotoxins, and sialic acid in the flow-through were detected by electrophoresis.

[0247] The endotoxin removal results are shown in Tables 9 and 10. The Diamond Mix-A Mustang and Phenyl 6FFhighSub-HS chromatography media showed excellent endotoxin removal effects, with a removal rate of over 98%.

[0248] Nucleic acid removal effect Figure 1 As shown, no obvious nucleic acid bands were observed in the purified glycosyl group after Diamond Mix-A Mustang purification, which is significantly better than other chromatography media.

[0249] Protein removal effect Figure 2As shown, no obvious protein bands were observed in the purified saccharides after purification with Diamond Mix-A Mustang, hydroxyapatite, and Monomix MC60.

[0250] In summary, considering the effectiveness of endotoxin, nucleic acid, and protein removal, as well as its criticality to the process, Diamond Mix-AMustang is the optimal chromatography medium.

[0251] Table 9. Effects of different chromatography media on endotoxin removal

[0252]

[0253] Table 10 Comprehensive Evaluation of Different Chromatographic Media

[0254]

[0255] Example 8 Preparation of purified capsular polysaccharides

[0256] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a final concentration of 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. The mixture was centrifuged three times repeatedly at 14000 rpm using a continuous flow centrifuge, and the supernatant was collected. The centrifuged supernatant was concentrated to 1 / 5 of the fermentation broth volume using a 100 kDa hollow fiber filter, and then replaced with purified water at a volume ratio of 1:9 four times, collecting the concentrated replacement retentate. The concentrated replacement collection was filtered through a 0.2 μm capsule filter to collect the crude sugar solution.

[0257] The crude sugar solution was adjusted with 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 2.75 mol / L NaCl, with a sample-to-adjustment buffer volume ratio of 4:1. After adjustment, the sample was loaded onto a Diamond MIX-A Mustang column (equilibrated with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, equilibrated for 3 CVs). The column was washed with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl for 2 CVs, and the flow-through was collected.

[0258] The flow-through solution was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, and then replaced four times with purified water at a volume ratio of 1:9. After the replacement was completed, it was filtered through a 0.2μm capsule filter.

[0259] After filtration, the sample was conditioned with 0.1 mol / L Tris-HCl buffer containing 1.65 mol / L NaCl, with a sample-to-conditioning buffer volume ratio of 4:1. After conditioning, the sample was loaded onto a Diamond MIX-A Mustang column and eluted for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl. Then, it was eluted for 3 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The eluent was collected at the peak.

[0260] The eluent was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, then four times with purified water at a volume ratio of 1:9. After replacement, the solution was filtered through a 0.2μm capsule filter. The filtered refined saccharide solution was then lyophilized to obtain purified polysaccharides.

[0261] Example 9 Preparation of purified capsular polysaccharides

[0262] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. It was then diluted 2-fold with a final concentration of 2.0 mol / L sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 with acetic acid, and stirred at 900–1500 rpm for 1 hour. The mixture was centrifuged three times repeatedly at 14000 rpm using a continuous flow centrifuge, and the supernatant was collected. The centrifuged supernatant was concentrated to 1 / 5 of the fermentation broth volume using a 100 kDa hollow fiber filter, and then replaced with purified water at a volume ratio of 1:9 four times, collecting the concentrated replacement retentate. The concentrated replacement collection was filtered through a 0.2 μm capsule filter to collect the crude sugar solution.

[0263] The crude sugar solution was adjusted with 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 2.75 mol / L NaCl, with a sample-to-adjustment buffer volume ratio of 4:1. After adjustment, the sample was loaded onto a Diamond MIX-A Mustang column (equilibrated with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, equilibrated for 3 CVs). The column was washed with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl for 2 CVs, and the flow-through was collected.

[0264] The flow-through solution was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, and then replaced four times with purified water at a volume ratio of 1:9. After the replacement was completed, it was filtered through a 0.2μm capsule filter.

[0265] After filtration, the sample was adjusted with 0.1 mol / L Tris-HCl (pH 8.0) buffer containing 2.75 mol / L NaCl, with a sample to adjustment buffer volume ratio of 4:1. After adjustment, the sample was loaded onto a Diamond MIX-A Mustang chromatography column and washed for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The flow-through was then collected.

[0266] The flow-through solution was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, then four times with purified water at a volume ratio of 1:9. After replacement, the solution was filtered through a 0.2μm capsule filter. The filtered refined saccharide solution was then lyophilized to obtain purified polysaccharide.

[0267] Example 10 Preparation of purified capsular polysaccharides

[0268] The fermentation broth obtained in Example 1 was collected and sterilized overnight with 0.5% (v / v) formaldehyde, maintaining pH 7.2 for >12 hours. The broth was centrifuged at 11000 rpm for 1 hour using an angle rotor centrifuge, and the supernatant was collected. The supernatant was diluted 2-fold with a 0.85% (w / v) sodium chloride solution, and 4.5% DOC solution was added to a final concentration of 0.15% (w / v), maintaining pH 7.3 ± 0.10, and stirred at 900–1500 rpm for 1 hour. The pH was adjusted to 4.0 ± 0.05 using acetic acid, and stirred at 900–1500 rpm for 1 hour. The broth was centrifuged at 11000 rpm for 1 hour, and the supernatant was collected. The centrifuged supernatant was concentrated to 1 / 5 of the fermentation broth volume using a 100KD hollow fiber filter, and then replaced with purified water at a volume ratio of 1:4, four times. The concentrated replacement retentate was collected. The concentrated replacement collection solution was filtered using a 0.2 μm capsule filter to collect the crude sugar solution.

[0269] The crude sugar solution was adjusted with 0.1 mol / L Tris-HCl buffer containing 1.65 mol / L NaCl, with a sample-to-adjustment buffer volume ratio of 4:1. After adjustment, the sample was loaded onto a Diamond MIX-A Mustang column (equilibrated with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, and equilibrated for 3 CVs). The column was eluted for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl, and then eluted for 3 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The eluent was collected at the peak.

[0270] The eluent was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, and then replaced four times with purified water at a volume ratio of 1:9. After the replacement was completed, it was filtered through a 0.2μm capsule filter.

[0271] After filtration, the sample was conditioned with 0.1 mol / L Tris-HCl buffer containing 1.65 mol / L NaCl, with a sample-to-conditioning buffer volume ratio of 4:1. After conditioning, the sample was loaded onto a Diamond MIX-A Mustang column and eluted for 2 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.33 mol / L NaCl. Then, it was eluted for 3 CVs with 0.02 mol / L Tris-HCl (pH 8.0) buffer containing 0.55 mol / L NaCl. The eluent was collected at the peak.

[0272] The eluent was concentrated and replaced using a 10KD membrane. First, it was replaced twice with 0.85% NaCl solution at a volume ratio of 1:9, then four times with purified water at a volume ratio of 1:9. After replacement, the solution was filtered through a 0.2μm capsule filter. The filtered refined saccharide solution was then lyophilized to obtain purified polysaccharides.

[0273] The yields of MenC purified capsular polysaccharide nucleic acid, protein, sialic acid, endotoxin and refined saccharide prepared in Examples 8-10 above are shown in Table 11. The refined saccharide prepared in Example 10 by angle rotor centrifugation and continuous two-step elution chromatography has better quality.

[0274] Table 11 Refined Sugar Yield and Test Results

[0275]

[0276] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A purified group C meningococcus ( Neisseria meningitidis The method for using capsular polysaccharides is characterized by, The method includes two steps: crude purification and purification. Purification is performed using either a two-step elution purification process or a flow-through combined elution purification process. The method satisfies one or more of the following: 1) Use a continuous flow centrifuge or an angle rotor centrifuge for coarse processing; 2) The buffer solution used for purification contains Tris-HCl and NaCl; and, 3) Purification was performed using a multi-mode strong anion exchange chromatography column.

2. The method as described in claim 1, characterized in that, For the initial processing, use a continuous flow centrifuge at 10,000-16,000 rpm; or, use an angle rotor centrifuge at 9,000-13,000 rpm. And / or, in the purification process, the multimode strong anion exchange chromatography column includes Diamond MIX-A Mustang, CaptoAdhere, Toyopearl MX-Trp-650M, POROS XQ and MEP HyperCel chromatography columns; Preferably, centrifugation is performed 2-4 times using a continuous flow centrifuge; more preferably, centrifugation is performed 3 times using a continuous flow centrifuge at 14,000 rpm. Alternatively, centrifuge for 0.5-1.5 hours using an angle rotor centrifuge; preferably, centrifuge at 11,000 rpm for 1 hour.

3. The method as described in claim 1 or 2, characterized in that, The "two-step elution and purification" includes a first-step purification and a second-step purification, which respectively include sample conditioning, sample loading, rinsing, elution, buffer system replacement and filtration. Preferably, the method satisfies one or more of the following: 1) The buffer solution used for sample conditioning contains 0.05-0.15 mol / L Tris-HCl and 1.5-1.8 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 1.65 mol / L NaCl. 2) Before loading the sample, a column equilibration step is also included. The equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl. 3) The rinsing buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl. 4) The elution buffer contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl. 5) The buffer system is replaced with a membrane pack for concentration, preferably a 3-30 kDa membrane pack; 6) The buffer system is first replaced with NaCl solution, then with pure water. The NaCl solution is preferably a 0.7%-1.0% NaCl solution. The volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9). 7) Use a filter membrane for filtration, preferably a filter membrane with a diameter of less than 0.3 μm; More preferably, the method satisfies one or more of the following: 1) During sample preparation, the volume ratio of crude sample to buffer solution is (3-5):1, for example, 4:1; 2) During equilibration, use buffer solution to equilibrate at 2-3 CV, preferably 3 CV; 3) During rinsing, use buffer solution to rinse 2-3 times per volume (CV), preferably 2 times per volume (CV); 4) During elution, use buffer to elute 2-3 CV, preferably 3 CV; 5) In the replacement of the buffer system, a 10KD membrane is used; 6) In the buffer system replacement, NaCl solution is used for 1-3 replacements, preferably 2; pure water is used for 3-5 replacements, preferably 4; and, 7) Use a filter membrane with a diameter of less than 0.23 μm for filtration.

4. The method as described in claim 1 or 2, characterized in that, The "flow-through combined elution purification" includes a first purification step and a second purification step. The first purification step includes sample conditioning, sample loading, flow-through collection, buffer system replacement and filtration. The second purification step includes sample conditioning, sample loading, rinsing, elution, buffer system replacement and filtration.

5. The method as described in claim 4, characterized in that, The first step of purification satisfies one or more of the following: 1) During sample preparation, the buffer solution used contains 0.05-0.15 mol / L Tris-HCl and 2.5-3.0 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 2.75 mol / L NaCl. 2) Before loading the sample, a column equilibration step is also included. The equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl. 3) In flow-through collection, the buffer solution used contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl. 4) In the replacement of the buffer system, membrane packs are used for concentration and replacement, preferably 3-30KD membrane packs; 5) In the buffer system replacement, NaCl solution is used first, followed by pure water. The NaCl solution is preferably a 0.7%-1.0% NaCl solution. The volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9). 6) In filtration, a filter membrane with a diameter of less than 0.3 μm is preferably used; Preferably, the first step of purification satisfies one or more of the following: 1) During sample preparation, the volume ratio of the sample to be purified to the buffer solution is (3-5):1, for example, 4:1; 2) During equilibration, use buffer solution to equilibrate at 2-3 CV, preferably 3 CV; 3) During flow-through collection, rinse with buffer for 2-3 cycles, preferably 2 cycles; 4) In the replacement of the buffer system, a 10KD membrane is used; 5) In the buffer system replacement, NaCl solution is used for 1-3 replacements, preferably 2; pure water is used for 3-5 replacements, preferably 4; and, 6) Use a filter membrane with a diameter of less than 0.23 μm for filtration.

6. The method as described in claim 4 or 5, characterized in that, The second purification step satisfies one or more of the following: 1) During sample preparation, the buffer solution used contains 0.05-0.15 mol / L Tris-HCl and 1.5-1.8 mol / L NaCl; for example, it contains 0.1 mol / L Tris-HCl and 1.65 mol / L NaCl. 2) Before loading the sample, a column equilibration step is also included. The equilibration buffer contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl. 3) During rinsing, the buffer solution used contains 0.01-0.03 mol / L Tris-HCl and 0.2-0.5 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.33 mol / L NaCl. 4) During elution, the buffer solution used contains 0.01-0.03 mol / L Tris-HCl and 0.4-0.7 mol / L NaCl; for example, it contains 0.02 mol / L Tris-HCl and 0.55 mol / L NaCl. 5) In the replacement of the buffer system, membrane concentration is used for concentration and replacement, preferably using 3-30KD membrane concentration; 6) In the buffer system replacement, NaCl solution is used first, followed by pure water. The NaCl solution is preferably a 0.7%-1.0% NaCl solution. The volume ratio of the solution to be replaced to the replacement solution is, for example, (1-2):(8-9). 7) In filtration, a filter membrane is used, preferably a filter membrane with a diameter of less than 0.3 μm; Preferably, the second purification step satisfies one or more of the following: 1) During sample preparation, the volume ratio of the sample to be purified to the buffer solution is (3-5):1, for example, 4:1; 2) During equilibration, use buffer solution to equilibrate at 2-3 CV, preferably 3 CV; 3) Use buffer solution to rinse for 2-3 cycles, preferably 2 cycles; 4) Elution with buffer solution for 2-3 CV, preferably 3 CV; 5) The buffer system was concentrated and replaced using a 10KD membrane pack. 6) In the buffer system, use NaCl solution for 1-3 displacements, preferably 2 displacements; use pure water for 3-5 displacements, preferably 4 displacements; and, 7) Use a filter membrane with a diameter of less than 0.23 μm for filtration.

7. The method according to any one of claims 1-6, characterized in that, The crude process includes the steps of collecting the supernatant after centrifugation in a continuous flow centrifuge or an angle rotor centrifuge, followed by concentration and replacement. Preferably, the method satisfies one or more of the following: 1) Use hollow fiber equipment for concentration, preferably using 80-120KD hollow fiber equipment, such as using 100KD hollow fiber equipment; 2) Use pure water for displacement, with a volume ratio of the solution to be replaced to the replacement solution of (1-2):(8-9), for example, 1:9; and, 3) Replace the water 3-5 times, preferably 4 times; More preferably, the concentration and replacement process also includes a filtration step, preferably using a filter membrane with a diameter of less than 0.3 μm, such as a filter membrane with a diameter of less than 0.23 μm.

8. The method according to any one of claims 1-7, characterized in that, The crude processing method uses fermentation broth and also includes the steps of cell lysis and release of capsular polysaccharides. Wherein, the roughing process satisfies one or more of the following: 1) Cells are lysed and capsular polysaccharides are released by adding DOC; 2) Before cell lysis, the fermentation broth also includes sterilization and dilution steps, such as sterilization with formaldehyde or dilution with sodium chloride solution; and, 3) After the release of capsular polysaccharides, a pH adjustment step is also included, for example, using acetic acid for pH adjustment.

9. A method for preparing a polysaccharide from the capsular region of group C meningococcus, characterized in that, The preparation method includes purifying group C meningococcal capsular polysaccharide using the method described in any one of claims 1-8; Preferably, the preparation method further includes fermentation culture of group C meningococci ( Neisseria meningitidis The steps include, preferably, starting the culture with seed culture, and / or, the culture process also includes a step of adding feed; More preferably, the feed comprises a feed medium, an alkaline solution, and a glucose solution.

10. The preparation method according to claim 9, characterized in that, The culture medium for culturing group C meningococci contains soybean peptone or yeast extract, glucose, sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate, ammonium sulfate, monosodium glutamate, and coenzyme I; and / or, the fed culture medium contains soybean peptone or yeast extract, coenzyme I, an amino acid mixture, a vitamin mixture, and T solution; Preferably, the amino acid mixture comprises UDP, arginine, cysteine, and glutamine; And / or, the vitamin mixture contains biotin, folic acid, vitamin B6, vitamin B1, riboflavin, nicotinic acid, niacin, calcium pantothenate and vitamin B12; And / or, the T solution contains MgSO4·7H2O, MnSO4·H2O, FeSO4·7H2O, CoCl·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3, NaMoO4·2H2O, and NiSO4·6H2O.

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