Streptococcus agalactiae capsular polysaccharide and purification method thereof

Abstract

The invention discloses streptococcus agalactiae capsular polysaccharide and a purification method thereof. The purification method comprises the following steps: sodium deoxycholate-calcium chloride pretreatment, ultrafiltration liquid exchange, chromatography by series strong anion exchange chromatography and weak anion exchange chromatography, ultrafiltration desalination, polysaccharide precipitation, sugar washing and freeze-drying. The method provided by the invention eliminates the use of phenol in the traditional capsular polysaccharide purification process, and reduces the harm to the environment and personnel. Carrying out crude sugar pretreatment by using a sodium deoxycholate treatment calcium chloride precipitation mode so as to preliminarily remove protein nucleic acid impurities; the flow-through mode chromatography of the chromatographic column is matched with tangential flow ultrafiltration desalination, so that the impurity content is lower, and the flow-through mode is used for impurity removal, so that the production amplification is more feasible; the strong anion and weak anion chromatography mode combination is matched with the organic solvent gradient sugar washing, so that polysaccharide pigment removal and endotoxin removal are facilitated; after the polysaccharide is precipitated, the polysaccharide is washed, homogenized, dispersed and freeze-dried, so that the polysaccharide freeze-dried powder with extremely low water content and uniform dispersion can be obtained, is convenient to take and use and is more stable to store.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a Streptococcus agalactiae capsular polysaccharide and a purification method thereof. Background Art

[0002] Streptococcus agalactiae is the main pathogen of neonatal infectious diseases, which can cause neonatal pneumonia, sepsis and meningitis, and is also one of the important factors leading to neonatal death. At the same time, Streptococcus agalactiae can also cause sepsis and skin infections in pregnant women, reduce the body's resistance, and cause infectious diseases in adults. The pathogenicity of Streptococcus agalactiae is mainly related to capsular polysaccharide antigens, etc. At present, there is a purification process to prepare Streptococcus agalactiae capsular polysaccharide for the preparation of vaccines to control the occurrence of Streptococcus agalactiae diseases.

[0003] Most of the existing capsular polysaccharide purification technologies are phenol extraction technologies, which use phenol to denature and precipitate proteins in crude sugar, remove proteins by centrifugation, and then use multi-step graded ethanol precipitation to remove nucleic acids and precipitate refined sugars. The polysaccharide is directly freeze-dried to achieve the purpose of purifying and harvesting capsular polysaccharides. However, the disadvantages are: 1) Human hazard: phenol is a toxic chemical substance with multiple potential hazards to the human body and the environment. The production and use of high-concentration phenol will affect or damage human skin, eyes, respiratory system, central nervous system, liver and kidneys, and long-term contact is carcinogenic and mutagenic; 2) Environmental hazard: It is toxic to aquatic organisms, and leakage will pollute the soil and water sources; 3) In the polysaccharide purification process, since phenol is not completely insoluble in water, some polysaccharides are dissolved in phenol and lost during the extraction process, and since phenol extraction is repeated many times, the loss of polysaccharides with high protein content is large, and the recovery rate is low; 4) It is easy to cause phenol residues in the final product to affect the quality; 5) The polysaccharide purification process using phenol extraction is complicated and time-consuming.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide a capsular polysaccharide of Streptococcus agalactiae and a purification method thereof, which eliminates the use of phenol in the traditional capsular polysaccharide purification process, reduces the harm to the environment and personnel, and can purify the polysaccharide quickly and efficiently.

[0006] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0007] A method for purifying Streptococcus agalactiae capsular polysaccharide comprises the following steps:

[0008] S1. Pretreatment: Dissolve the raw sugar in buffer, add sodium deoxycholate, stir and mix, then add calcium chloride, incubate for a certain period of time, centrifuge and take the supernatant;

[0009] S2, ultrafiltration and liquid replacement: ultrafiltration and liquid replacement of the supernatant obtained in S1;

[0010] S3, chromatography: chromatograph the solution obtained in S2 using a series of strong anion exchange chromatography and weak anion exchange chromatography, and collect the chromatographic solution;

[0011] S4, ultrafiltration desalination: ultrafiltration and concentration of the chromatographic solution obtained in S3 to obtain a polysaccharide concentrate;

[0012] S5. Polysaccharide precipitation: using a precipitant to precipitate the polysaccharide concentrate, then centrifuging and collecting the precipitate;

[0013] S6, washing sugar and freeze-drying: washing the precipitate obtained in S5, and then freeze-drying to obtain the capsular polysaccharide of Streptococcus agalactiae.

[0014] In one or more embodiments of the present invention: in step S1, sodium deoxycholate is added to the solution until the mass fraction of sodium deoxycholate in the solution is 0.1-1%.

[0015] In one or more embodiments of the present invention, in step S1, calcium chloride solution is added to the solution until the concentration of calcium chloride in the solution is 0.25-1 mol / L.

[0016] In one or more embodiments of the present invention, in step S2, ultrafiltration is performed using a tangential flow method, and the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is 10 to 50 kDa.

[0017] In one or more embodiments of the present invention, in step S3, the strong anion exchange chromatography medium is Captoadhere, and the weak anion exchange chromatography medium is Capto DEAE.

[0018] In one or more embodiments of the present invention, in step S4, ultrafiltration is performed using a tangential flow method, and the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is 10 to 50 kDa.

[0019] In one or more embodiments of the present invention, in step S5, the precipitant is sodium chloride and ethanol, sodium chloride solution is added to the polysaccharide concentrate to a final sodium chloride concentration of 0.3-1.0 mol / L, ethanol is added to a final ethanol concentration of 70-80%, the mixture is stirred and mixed, and allowed to stand at 2°C-8°C for 12-24 hours.

[0020] In one or more embodiments of the present invention, the washing operation in step S6 is:

[0021] S61, mixing the precipitate with anhydrous ethanol in a mass ratio of 1:(2-10), homogenizing, dispersing, centrifuging, and collecting the precipitate;

[0022] S62, mixing the S61 precipitate with acetone in a mass ratio of 1: (2-10), homogenizing, dispersing, centrifuging, and collecting the precipitate; repeating this step several times;

[0023] S63. Mix the precipitate obtained in S62 with diethyl ether in a mass ratio of 1:(2-10), homogenize, disperse, centrifuge, and collect the precipitate.

[0024] In one or more embodiments of the present invention, the freeze-drying operation in step S6 is: pressure below 80 Pa, temperature -35 to -60°C, and time 12 to 48 hours.

[0025] Another specific embodiment of the present invention provides a technical solution as follows:

[0026] A Streptococcus agalactiae capsular polysaccharide is prepared by the Streptococcus agalactiae capsular polysaccharide purification method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention uses sodium deoxycholate as a raw sugar pretreatment reagent. Since desalted sodium cholate can be used as a bactericide in the fermentation sterilization process, the use of desalted sodium cholate for pretreatment does not introduce new impurities, which can be removed uniformly later;

[0029] (2) The present invention uses Capto adhere composite mode in series with Capto DEAE chromatography as the main impurity removal method, eliminating the use of phenol in the traditional capsular polysaccharide purification process, reducing the harm to the environment and personnel; the chromatography mode used at the same time is the flow-through mode, combined with sodium deoxycholate-calcium chloride impurity pretreatment to optimize the loading capacity, which can purify the polysaccharide solution in large quantities, quickly and efficiently;

[0030] (3) Using tangential flow ultrafiltration to exchange the solution and concentrate the polysaccharide solution for polysaccharide precipitation can reduce the amount of ethanol used;

[0031] (4) Since polysaccharides are highly viscous, they are easily lost or inconvenient to use in subsequent process stages due to viscosity problems. Using organic solvent gradient sugar washing and cooperating with a homogenizer can effectively dehydrate and evenly disperse the refined sugar. After freeze-drying, a uniform, dispersed, and extremely low-water refined sugar powder can be obtained, which is easy to use in subsequent process stages.

[0032] (5) The combination of strong anion and weak anion chromatography modes with organic solvent gradient washing results in excellent polysaccharide pigment and endotoxin removal effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0034] Figure 1 The chromatogram in Example 3 of the present invention;

[0035] Figure 2 This is the chromatographic peak diagram in Example 4 of the present invention. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0037] A specific embodiment of the present invention provides a method for purifying capsular polysaccharide of Streptococcus agalactiae, comprising steps 1-6.

[0038] Step 1, pretreatment: dissolve the raw sugar in buffer, add sodium deoxycholate, stir to mix, then add calcium salt, incubate for a certain period of time, centrifuge, and take the supernatant.

[0039] Specifically, the raw sugar is dissolved in a sodium acetate buffer to prepare a polysaccharide solution, the sodium acetate buffer concentration is 300 mmol / L, the pH is 6.7-6.9, and the raw sugar concentration in the polysaccharide solution is 5-30 mg / ml, preferably 10-20 mg / ml. A 5% sodium deoxycholate aqueous solution is added to the polysaccharide solution until the mass fraction of sodium deoxycholate in the polysaccharide solution is 0.1-1%, preferably 0.5%, and stirred for 10-30 minutes to mix. After adding the sodium deoxycholate aqueous solution, the raw sugar concentration in the polysaccharide solution is 1-30 mg / ml, preferably 5-10 mg / ml. Then add calcium chloride aqueous solution to the polysaccharide solution until the calcium chloride concentration in the polysaccharide solution is 0.25-1 mol / L, preferably 0.5 mol / L, and then place it in a refrigerator at 2-8°C for incubation for more than 3 hours, and then centrifuge it with a centrifugal force parameter of 10000g and a centrifugal time of 10-30 minutes.

[0040] Sodium deoxycholate (DOC) is used as an anionic surfactant to dissolve proteins. Calcium chloride is used as a precipitant under acidic conditions. Divalent cation calcium ions can precipitate sodium deoxycholate, and calcium chloride can chelate some nucleic acids, thereby removing some proteins and nucleic acids from crude sugar by co-precipitation.

[0041] Step 2, ultrafiltration and liquid replacement: the supernatant obtained in S1 is subjected to ultrafiltration and liquid replacement.

[0042] Specifically, the supernatant after centrifugation in step 1 is collected and ultrafiltered with injection water. The ultrafiltration method is equal volume ultrafiltration and the ultrafiltration multiple is 10 times. A tangential flow ultrafiltration membrane package is used for ultrafiltration. The molecular weight cutoff of the tangential flow ultrafiltration membrane package is 10 to 50 kDa, preferably 10 kDa, and the material of the tangential flow ultrafiltration membrane package is preferably regenerated cellulose. Then the polysaccharide solution is concentrated to a polysaccharide dissolved concentration of 1 to 20 mg / ml.

[0043] The ultrafiltered polysaccharide solution was diluted 2 times with a 2-fold concentration flow-through buffer, and the pH was adjusted to 7.5-8.0. The 2-fold concentration flow-through buffer was: 100 mmol / L phosphate buffer, 300 mmol / L sodium chloride, pH 7.5-8.0.

[0044] Step 3, chromatography: chromatograph the filtrate obtained from S2 using a series of strong anion exchange chromatography and weak anion exchange chromatography, and collect the chromatographic solution.

[0045] Specifically, a 1-fold concentration flow-through buffer is used for loading, and the 1-fold concentration flow-through buffer is: 50 mmol / L phosphate buffer, 150 mmol / L sodium chloride, pH 7.5-8.0. Capto adhere is used as a strong anion exchange chromatography medium, and Capto DEAE is used as a weak anion exchange chromatography medium. The ratio of the series length of the two is (2-1):1, preferably 2:1, the total column height is 5-80 cm, preferably 15-30 cm; the loading amount is 0.5-10 CV, and the flow rate is 60-300 cm / h, preferably 90-150 cm / h.

[0046] Use UV206nm as the detection wavelength of the UV detector, use UV206nm peak as the collection judgment condition, start collecting the flow-through chromatographic liquid when UV206 peaks, and stop collecting the flow-through liquid when the UV 280nm wavelength begins to peak. Combining strong anion exchange chromatography with weak anion exchange chromatography and subsequent organic gradient sugar washing can achieve excellent polysaccharide pigment removal and endotoxin removal effects.

[0047] Step 4, ultrafiltration desalting: ultrafiltration and concentration of the chromatographic solution obtained in S3 to obtain a polysaccharide concentrate.

[0048] Specifically, a NaCl solution with a concentration of 0.3M and a pH of 7.0 is used for ultrafiltration, the ultrafiltration method is equal volume ultrafiltration, the ultrafiltration multiple is 10 times, and a tangential flow ultrafiltration membrane package is used for ultrafiltration, the molecular weight cutoff of the tangential flow ultrafiltration membrane package is 10 to 50 kDa, preferably 10 kDa, and the material of the tangential flow ultrafiltration membrane package is preferably regenerated cellulose. Finally, the polysaccharide solution is concentrated to 2 times the polysaccharide dissolved concentration.

[0049] Step 5, polysaccharide precipitation: the polysaccharide concentrate is precipitated using a precipitant, and then centrifuged to collect the precipitate.

[0050] Specifically, a sodium chloride aqueous solution with a concentration of 4 mol / L is added to the polysaccharide concentrate until the final sodium chloride concentration in the polysaccharide solution is 0.3-1.0 mol / L, and then ethanol with a concentration of 95% is added until the final ethanol concentration in the polysaccharide solution is 70-80%, preferably 75%. Stir for 30 minutes, then put it in a refrigerator at 2°C-8°C for 12-24 hours, and then collect the precipitated polysaccharide by centrifugation, the centrifugal force parameter is 10000g, the centrifugal time is 10-30 minutes, and the centrifugal temperature is 4°C-8°C.

[0051] Step 6, washing sugar and freeze-drying: washing the precipitate obtained in S5, and then freeze-drying to obtain the capsular polysaccharide of Streptococcus agalactiae.

[0052] Specifically, the polysaccharide precipitate is added to anhydrous ethanol for washing, and the mass ratio of the polysaccharide precipitate to the anhydrous ethanol is 1: (2-10), and the preferred mass ratio is 1: (4-6). A homogenizer is used to break up and disperse the polysaccharide precipitate, and then the dispersed precipitated polysaccharide is collected by centrifugation, the centrifugal force parameter is 10000g, the centrifugal time is 15min, and the centrifugal temperature is 4°C. Then the separated precipitated polysaccharide is mixed with acetone at a mass ratio of 1: (2-10), and the polysaccharide precipitate is also broken up and dispersed by a homogenizer, and then the dispersed precipitated polysaccharide is collected by centrifugation, the centrifugal force parameter is 10000g, the centrifugal time is 15min, and the centrifugal temperature is 4°C, and the polysaccharide precipitate is washed with acetone twice. Finally, the collected polysaccharide precipitate is mixed with ether at a mass ratio of 1: (2-10), and the polysaccharide precipitate is also broken up and dispersed by a homogenizer, and then the dispersed precipitated polysaccharide is collected by centrifugation, the centrifugal force parameter is 10000g, the centrifugal time is 15min, and the centrifugal temperature is 4°C. The washed polysaccharide precipitate is freeze-dried to obtain powdered Streptococcus agalactiae capsular polysaccharide. In the freeze-drying stage, the pressure is maintained below 80 Pa, the temperature is -35°C to -60°C, and the freeze-drying time is 12 to 48 hours.

[0053] Since polysaccharides are highly viscous, they can easily be lost or inconvenient to use in subsequent process stages. Using organic solvent gradient sugar washing and cooperating with a homogenizer can effectively dehydrate the refined sugar and disperse it evenly. After freeze-drying, a uniform, dispersed, and extremely low-water refined sugar powder can be obtained, which is easy to use in subsequent process stages.

[0054] Another specific embodiment of the present invention provides a Streptococcus agalactiae capsular polysaccharide, which is prepared by the above-mentioned Streptococcus agalactiae capsular polysaccharide purification method.

[0055] The present invention is further described in detail below in conjunction with specific embodiments.

[0056] The GBSⅡ crude sugar used in the present invention has a total protein content of 5.50%, a total nucleic acid content of 2.24%, and a total polysaccharide content of 16.50%; the GBS Ia crude sugar used has a total protein content of 4.01%, a total nucleic acid content of 8.27%, and a total polysaccharide content of 11.20%.

[0057] Type Ⅱ Raw Sugar DOC Pretreatment

[0058] Example 1

[0059] 1) Dissolving crude sugar: Dissolve GBSⅡ crude sugar in 0.3M sodium acetate buffer to obtain a crude sugar solution with a pH of 6.70 and a crude sugar concentration of 10 mg / ml;

[0060] 2) DOC treatment: add 1% DOC aqueous solution to the raw sugar solution until the final DOC concentration in the solution is 0.5% and the final raw sugar concentration is 5 mg / ml, and stir for 20 min;

[0061] 3) Calcium chloride precipitation incubation: add 3M calcium chloride aqueous solution to the raw sugar solution until the final concentration of calcium chloride in the solution is 0.5M, and then place it at 6.5°C for precipitation incubation for 3 hours;

[0062] 4) Centrifugation to remove precipitate: Use a centrifuge to centrifuge at 10,000 g for 30 min at 6° C. Collect the supernatant and filter the supernatant using a 0.2-1.0 μm filter.

[0063] Example 2

[0064] 1) Dissolution of crude sugar: Dissolve GBSⅡ crude sugar in 0.3M sodium acetate buffer to obtain polysaccharide solution. The pH of the solution is 6.85 and the crude sugar concentration is 10 mg / ml.

[0065] 2) DOC treatment: add 5% DOC aqueous solution to the raw sugar solution until the final DOC concentration in the solution is 0.5% and the final raw sugar concentration is 5 mg / ml, and stir for 20 min;

[0066] 3) Calcium chloride precipitation incubation: add 1 M calcium chloride aqueous solution to the raw sugar solution until the final concentration of calcium chloride in the solution is 0.5 M, and then place it at 6.5°C for precipitation incubation for 3 hours;

[0067] 4) Centrifugation to remove precipitate: Use a centrifuge to centrifuge at 10,000 g for 20 min at 4° C. Collect the supernatant and filter the supernatant using a 0.2-1.0 μm filter.

[0068] The DOC pretreatment results in Example 1 and Example 2 are shown in Table 1:

[0069] Table 1 DOC pretreatment results in Example 1 and Example 2

[0070] project Protein removal rate Nucleic acid removal rate Polysaccharide recovery rate Example 1 47.14％ 45.69％ 89.39％ Example 2 59.84％ 51.99％ 87.55％

[0071] As can be seen from Table 1, pre-treatment of crude sugar with sodium deoxycholate and calcium chloride can remove about 50% of proteins and nucleic acids while ensuring the recovery rate of polysaccharides, and can optimize the subsequent chromatography loading capacity.

[0072] Type II and Type Ia crude sugar chromatography

[0073] Example 3

[0074] 1) Dissolution of crude sugar: Dissolve GBSⅡ crude sugar in chromatography flow-through buffer, the flow-through buffer concentration is: 50mmol / L phosphate buffer, 150mmol / L sodium chloride, pH 8.0; the final concentration of crude sugar in the solution is 5mg / ml;

[0075] 2) Chromatography: The crude sugar solution was filtered using a 0.22 μm filter, and the filtrate was passed through a Capto adhere column connected in series with a Capto DEAE column. The length ratio of the chromatography column Capto adhere to Capto DEAE was 2:1, the total column height was 24 cm, the sample loading volume was 2 CV, and the flow rate was 100 cm / h.

[0076] The concentration of the loading buffer A was: 50mmol / L phosphate buffer, 150mmol / L sodium chloride, pH 8.0; the concentration of the elution buffer B was: 50mmol / L phosphate buffer, 1000mmol / L sodium chloride, pH 8.0;

[0077] The chromatographic liquid collection judgment condition is that the UV detector has a peak at 206nm wavelength. The chromatographic liquid starts to be collected when the UV206nm peak starts. When the UV206nm peak baseline stabilizes or the UV 280nm / 260nm wavelength starts to peak, the collection of the chromatographic liquid is stopped. Figure 1 shown.

[0078] Example 4

[0079] 1) Dissolving raw sugar: Dissolve GBS Ia raw sugar in chromatography flow-through buffer, the flow-through buffer concentration is: 50 mmol / L phosphate buffer, 150 mmol / L sodium chloride, pH 8.0; the final concentration of raw sugar in the solution is 5 mg / ml;

[0080] 2) Chromatography: The crude sugar solution was filtered using a 0.22 μm filter, and the filtrate was passed through a Capto adhere column connected in series with a Capto DEAE column. The length ratio of the chromatography column Capto adhere to Capto DEAE was 2:1, the total column height was 24 cm, the sample loading volume was 2 CV, and the flow rate was 100 cm / h.

[0081] The concentration of the loading buffer A was: 50mmol / L phosphate buffer, 150mmol / L sodium chloride, pH 8.0; the concentration of the elution buffer B was: 50mmol / L phosphate buffer, 1000mmol / L sodium chloride, pH 8.0;

[0082] The chromatographic liquid collection judgment condition is that the UV detector has a peak at 206nm wavelength. The chromatographic liquid starts to be collected when the UV206nm peak starts. When the UV206nm peak baseline stabilizes or the UV 280nm / 260nm wavelength starts to peak, the collection of the chromatographic liquid is stopped. Figure 2 Show.

[0083] The DOC pre-treatment results in Example 3 and Example 4 are shown in Table 2:

[0084] Table 2 DOC pretreatment results in Example 3 and Example 4

[0085] project Protein removal rate Nucleic acid removal rate Polysaccharide recovery rate Example 3 73.77％ 97.77％ 90.70％ Example 4 82.64％ 98.19％ 96.62％

[0086] Combination Figure 1 , Figure 2 From Table 2, it can be found that when the conditions disclosed in the present invention are used for polysaccharide flow-through mode chromatography, the chromatogram UV detector shows that the UV206 peak is completely separated from the UV280 and UV260 peaks. From the test results, it can be seen that the protein and nucleic acid residues are low and the polysaccharide recovery rate is extremely high.

[0087] Type II crude sugar purification process

[0088] Example 5

[0089] 1) Dissolution of crude sugar: Weigh GBSⅡ crude sugar and dissolve it in 0.3 M sodium acetate buffer. The pH of the solution is 6.70 and the polysaccharide concentration is 10 mg / mL.

[0090] 2) DOC-CaCl2 precipitation impurities:

[0091] DOC treatment: Add 5% DOC aqueous solution to the polysaccharide solution until the final DOC concentration in the solution is 0.5%, and stir for 15 minutes;

[0092] CaCl2 precipitation: Add 3M calcium chloride aqueous solution (pH 6.7±0.3) to the polysaccharide solution after DOC treatment until the final concentration of calcium chloride in the polysaccharide solution is 0.5M, and then place the polysaccharide solution in a 6.5℃ refrigerator for precipitation incubation for 3h. Then take out the polysaccharide solution, stir for 1min, and then centrifuge to remove the precipitate. The centrifugation parameters are 10000g, time is 30min, and temperature is 4℃. Collect the supernatant and clarify and filter using a 0.45μm filter cup.

[0093] 3) Ultrafiltration fluid replacement:

[0094] The filtrate was ultrafiltered using a Sartorius RC membrane with a molecular weight cutoff of 10 kDa, and integrity and water flux tests were performed before use;

[0095] The supernatant was concentrated to a sugar-soluble volume of 10 mg / ml, diluted 1-fold with water for injection, ultrafiltered 10 times or until the conductivity was stable and TMP was less than 0.8 bar during ultrafiltration, the filtrate was collected, and the volume was adjusted to a sugar-soluble volume of 10 mg / ml with water for injection;

[0096] After ultrafiltration, the sample was diluted twice with a 2-fold concentration of chromatography flow-through buffer, and the pH was adjusted to 8.0, and the conductivity was detected; the 2-fold concentration of chromatography flow-through buffer was: 100 mM PB, 300 mM NaCl, pH 8.0;

[0097] 4) Chromatography:

[0098] Column pretreatment: The chromatography column is a Capto adhere column connected in series with a Capto DEAE column, the length ratio of the chromatography column Capto adhere to Capto DEAE is 2:1, and the total column height is 24 cm. First, wash the column with a 0.5M sodium hydroxide solution for 60 minutes at a flow rate of 120 cm / h; then wash the column with a 1mol / L sodium chloride buffer solution, the volume of the sodium chloride buffer solution is not less than 3CV, and the flow rate is 120 cm / h; finally, balance with 10 CV of flow-through buffer until the baseline is stable, and the flow rate is 100 cm / h; the flow-through buffer is: 50mM PB, 150mM NaCl, pH8.0;

[0099] Sample loading: The concentration of the sample flow-through buffer A is: 50mmol / L phosphate buffer, 150mmol / L sodium chloride, pH 8.0; the concentration of the elution buffer B is: 50mmol / L phosphate buffer, 1000mmol / L sodium chloride, pH 8.0; the sample loading volume is 2CV, and the flow rate is 100cm / h;

[0100] After the peak appears, the flow-through peak is collected. The judgment condition for the collection of the chromatographic liquid is the peak of the UV detector at 206nm wavelength. The collection of the chromatographic flow-through liquid begins when the UV206nm peak appears. When the UV206nm peak baseline stabilizes or the UV 280nm / 260nm wavelength begins to peak, the collection of the flow-through chromatographic liquid is stopped.

[0101] 5) TFF liquid replacement: The flow-through peak sample was replaced with 0.3 M NaCl (pH 7.0) for 1-fold volume replacement, and then concentrated to 2-fold sugar concentration volume;

[0102] 6) Sugar precipitation: add a 4 mol / L sodium chloride solution to the sample until the final concentration of sodium chloride in the solution is 0.3 mol / L, then add 95% ethanol to the solution until the final concentration of ethanol in the solution is 75%, precipitate sugar, stir for 30 min, and then place in a 6.5°C refrigerator for 20 h;

[0103] 7) Sugar washing:

[0104] Centrifugal collection of polysaccharides: centrifuge at 10000g, 30min, 4°C to collect precipitated polysaccharides;

[0105] Sugar washing: Use anhydrous ethanol, acetone and ether washing solutions to wash the sugar in sequence, add the precipitated polysaccharide to anhydrous ethanol, the mass ratio of precipitated polysaccharide to anhydrous ethanol is 1:5, use a homogenizer to break and disperse, collect the dispersed precipitated polysaccharide by centrifugation, the centrifugation parameters are 10000g, 30min, 4°C, separate the precipitated polysaccharide; then repeat the above washing operation with acetone, and repeat the washing with acetone twice; finally, repeat the above washing operation with ether once for the precipitated polysaccharide; after the sugar washing is completed, place the precipitated polysaccharide in a biological safety cabinet and let it stand until the residual ether solution evaporates;

[0106] 8) Freeze drying: The freeze dryer was precooled at -40°C for 30 min, the pressure was set to 100 Pa, and then the precipitated polysaccharide was vacuum dried for 12 h.

[0107] Comparative Example 1

[0108] Traditional phenol extraction process:

[0109] 1) Sugar dissolution: Dissolve GBSⅡ crude sugar in 0.3 mol / L sodium acetate solution to 10 mg / ml, pH 7.0±0.1.

[0110] 2) Phenol extraction: Add the prepared phenol solution to the polysaccharide solution at a volume ratio of 1:0.5 (polysaccharide solution / phenol solution) and stir with a magnetic stirrer for 10 min. The mixed solution is divided into centrifuge cups and the supernatant is collected by centrifugation. Repeat the above operation for phenol extraction 5 times.

[0111] 3) Ultrafiltration to remove phenol: the supernatant was diluted with 20 times injection water, and concentrated to the original volume by ultrafiltration using a 10KD membrane package. Four times the volume of injection water was used for ultrafiltration each time, and the ultrafiltration was repeated 5 times.

[0112] 4) Precipitate nucleic acids: While stirring, slowly add 3 mol / L calcium chloride solution to the polysaccharide solution to make the final concentration reach 0.5 mol / L, then slowly add 95% ethanol to the theoretical final concentration of ethanol for precipitating nucleic acids to 25%, stir for 30 minutes, and place at 2°C for 15 hours after stirring.

[0113] 5) Removal of nucleic acids:

[0114] ① Centrifuge, collect the supernatant, clarify and filter, filter the pore size range (0.45μm~1.0μm), and collect the clarified and filtered polysaccharide solution;

[0115] ②Use the membrane package with a pore size of 10KD for ultrafiltration, and use 1 times the volume of injection water to ultrafilter the polysaccharide solution for 10 times in total. The last ultrafiltration is concentrated to a theoretical concentration of 40 mg / ml, and the ultrafiltered solution is collected.

[0116] 6) Precipitating polysaccharides: While stirring, slowly add 4 mol / L sodium chloride solution to the polysaccharide solution to make its final concentration reach 0.3 mol / L, then slowly add 95% ethanol to make the theoretical final concentration of precipitated polysaccharide ethanol reach 75%, stir for 30 minutes, and place in a medical drug storage box for more than 12 hours after stirring.

[0117] 7) Sugar washing: collect the precipitate by centrifugation; wash the sugar in the order of anhydrous ethanol-acetone-acetone-ether, and collect the precipitate by centrifugation at each step. Use a homogenizer to break and wash the sugar.

[0118] 8) Freeze drying: vacuum freeze drying at -45°C, with the drying time not less than 8 hours.

[0119] The purification results of Example 5 and Comparative Example 1 are shown in Table 3:

[0120] Table 3 Purification results of Example 5 and Comparative Example 1

[0121]

[0122] It can be seen from Table 3 that the various indicators of the refined sugar of Streptococcus agalactiae obtained by the present invention are good. Since the capsular polysaccharide of Streptococcus agalactiae has not yet been included in the national pharmacopoeia, the present invention refers to the pharmacopoeia indicators of the capsular polysaccharide of Pneumococcus. The protein and nucleic acid residues in the present invention can meet the standards, and the refined sugar yield is higher than that of the traditional phenol extraction process.

[0123] In summary, the present invention eliminates the use of phenol in the traditional capsular polysaccharide purification process, reducing the harm to the environment and personnel; the crude sugar is pretreated by a combination of sodium deoxycholate treatment and calcium chloride precipitation, so that protein and nucleic acid impurities are initially removed and the subsequent loading capacity is optimized; the chromatography column flow-through mode chromatography is combined with tangential flow ultrafiltration desalination to lower the impurity content, and the use of the flow-through mode as the main impurity removal process makes production scale-up more feasible. In addition, the combination of strong anion and weak anion chromatography modes and organic solvent gradient sugar washing makes the polysaccharide pigment removal and endotoxin removal effects excellent; after the polysaccharide precipitation, the sugar is washed and homogenized and then freeze-dried to obtain a polysaccharide freeze-dried powder with extremely low water content and uniform dispersion, which is easy to use and the refined sugar storage is more stable.

[0124] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0125] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for purifying capsular polysaccharide of Streptococcus agalactiae, characterized in that: The following steps are involved: S1. Pretreatment: Dissolve the raw sugar in buffer, add sodium deoxycholate, stir and mix, then add calcium chloride, incubate for a certain period of time, centrifuge and take the supernatant; S2, ultrafiltration and liquid replacement: ultrafiltration and liquid replacement of the supernatant obtained in S1; S3, chromatography: chromatograph the solution obtained in S2 using a series of strong anion exchange chromatography and weak anion exchange chromatography, and collect the chromatographic solution; S4, ultrafiltration desalting: ultrafiltration and concentration of the chromatographic solution obtained in S3 to obtain a polysaccharide concentrate; S5. Polysaccharide precipitation: using a precipitant to precipitate the polysaccharide concentrate, then centrifuging and collecting the precipitate; S6, washing sugar and freeze-drying: washing the precipitate obtained in S5, and then freeze-drying to obtain the capsular polysaccharide of Streptococcus agalactiae.

2. The method for purifying Streptococcus agalactiae capsular polysaccharide according to claim 1, characterized in that: In step S1, sodium deoxycholate is added to the solution until the mass fraction of sodium deoxycholate in the solution is 0.1-1%.

3. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: In step S1, a calcium chloride solution is added to the solution until the concentration of calcium chloride in the solution is 0.25 to 1 mol / L.

4. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: In step S2, ultrafiltration is performed using a tangential flow method, and the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is 10 to 50 kDa.

5. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: In step S3, Capto adhere is used as the strong anion exchange chromatography medium, and Capto DEAE is used as the weak anion exchange chromatography medium.

6. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: In step S4, ultrafiltration is performed using a tangential flow method, and the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration is 10 to 50 kDa.

7. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: In step S5, the precipitant is sodium chloride and ethanol. Sodium chloride solution is added to the polysaccharide concentrate to a final sodium chloride concentration of 0.3-1.0 mol / L, and ethanol is added to a final ethanol concentration of 70-80%. The mixture is stirred and mixed, and allowed to stand at 2°C-8°C for 12-24 hours.

8. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: The washing operation in step S6 is: S61, mixing the precipitate with anhydrous ethanol in a mass ratio of 1:(2-10), homogenizing, dispersing, centrifuging, and collecting the precipitate; S62, mixing the S61 precipitate with acetone in a mass ratio of 1: (2-10), homogenizing, dispersing, centrifuging, and collecting the precipitate; repeating this step several times; S63. Mix the precipitate obtained in S62 with diethyl ether in a mass ratio of 1:(2-10), homogenize, disperse, centrifuge, and collect the precipitate.

9. The method for purifying capsular polysaccharide of Streptococcus agalactiae according to claim 1, characterized in that: The freeze-drying operation in step S6 is as follows: the pressure is below 80 Pa, the temperature is -35 to -60°C, and the time is 12 to 48 hours.

10. A Streptococcus agalactiae capsular polysaccharide, characterized in that: The polysaccharide is prepared by the method for purifying the capsular polysaccharide of Streptococcus agalactiae according to any one of claims 1 to 9.

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