A process for purifying pneumococcal monovalent polysaccharides

Abstract

The application discloses a kind of purification process of pneumococcal monovalent polysaccharide, and is specifically related to the field of biological medicine.The process includes pretreatment, anion chromatography, composite chromatography and biomimetic ligand chromatography.The purification process of the application first uses acid precipitation process to remove 30% of protein and nucleic acid, then uses anion chromatography for crude extraction, removes more than 80% of protein and nucleic acid, and the anion chromatography crude extraction can handle large volume of feed liquid;Then use composite chromatography to purify, remove residual protein, nucleic acid and endotoxin, and further concentrate polysaccharide with high load;Finally, use biomimetic ligand chromatography to deeply remove impurities, target removal of trace nucleic acid, finally obtain single polysaccharide recovery rate is more than 90%, protein content is less than 0.1%, nucleic acid content is less than 0.1%, endotoxin content is less than 0.1 EU / mL, and removal rate reaches more than 99%.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a purification process for a monovalent polysaccharide from pneumococcus. Background Technology

[0002] Streptococcus pneumoniae is a Gram-positive bacterium with over 90 serotypes. Most Streptococcus pneumoniae bacteria are coated with a capsular polysaccharide. This capsular polysaccharide can evade recognition by the immune system and prevent phagocytosis by immune cells, thus allowing the bacteria to multiply and cause disease. Years of research and clinical trials have demonstrated that Streptococcus pneumoniae capsular polysaccharides, when used as vaccines, can induce specific antibodies and provide excellent immunoprotection against the corresponding Streptococcus pneumoniae strains. The composition and structure of capsular polysaccharides differ among different serotypes of Streptococcus pneumoniae; therefore, combinations of polysaccharides from different serotypes are typically used as vaccines to prevent the pathogenicity of major Streptococcus pneumoniae strains.

[0003] During the fermentation process of Streptococcus pneumoniae used to prepare polysaccharide vaccines, in addition to producing capsular polysaccharides, a large number of metabolites are also generated. In order to meet the purity requirements of the capsular polysaccharides for vaccine preparation, the fermentation broth usually has to undergo multiple purification steps to remove impurities such as proteins, nucleic acids, and endotoxins from the crude polysaccharide product, thereby avoiding possible adverse reactions caused by subsequent vaccination.

[0004] Currently, the commonly used method is ethanol precipitation, which can effectively remove protein contaminants from polysaccharides, but it is difficult to meet the purity requirements for injectable vaccines. In addition, the use of ethanol brings the following problems: 1) The use of ethanol requires fire prevention facilities, and the design of such facilities is very costly; 2) The amount of ethanol required is huge, almost 4-6L of ethanol is needed for every liter of processed material, and the waste liquid is very difficult to treat; 3) The polysaccharides after treatment are difficult to reconstitute, requiring steps such as freeze-drying.

[0005] Chinese patent CN112646050A describes a process for purifying pneumococcal polysaccharides using ion exchange chromatography. This patent uses ion exchange chromatography to purify pneumococcal polysaccharides, but the sample loading amount is low. Increasing the sample loading amount will significantly increase the content of protein and nucleic acid, resulting in a low removal rate. Although the low sample loading amount can ensure the removal rate, the processing capacity is insufficient, and the efficiency is too low.

[0006] Chinese patent CN107043431A discloses a method for purifying bacterial capsular polysaccharides. It uses a combination of ion exchange chromatography and hydroxyphosphate ash chromatography to obtain a purified capsular polysaccharide solution. However, when using the combination chromatography alone, the recovery rate of the product is only 60 to 80%, and the protein content is greater than 0.5%, indicating a low removal rate. Furthermore, this patent still faces the problem of processing small sample volumes. Summary of the Invention

[0007] Therefore, the present invention provides a purification process for pneumococcal monovalent polysaccharides to solve the problems in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] According to the present invention, a purification process for a monovalent polysaccharide of *Streptococcus pneumoniae* is provided, the process comprising:

[0010] Step 1, Pre-processing

[0011] After activation, expansion culture and fermentation culture in a fermenter, pneumococcus was inactivated and polysaccharides were harvested. The bacterial cells were removed by centrifugation, and the feed solution was concentrated by ultrafiltration. Acid precipitation was used to initially remove impurities including some proteins and some nucleic acids. The acid precipitation centrifuged liquid was pretreated by ultrafiltration system and then added to the chromatographic equilibrium system PBS to obtain the pretreated crude extract.

[0012] Step 2, Anion Chromatography

[0013] The pretreated crude extract was passed through an anion exchange chromatography column, and the feed solution was collected in flow-through mode to obtain DEAE flow-through solution. The solution was then switched to a low conductivity Tris buffer system for ultrafiltration to obtain DEAE treated solution.

[0014] Step 3, Composite Chromatography

[0015] The initial concentrated polysaccharide solution was loaded onto a composite chromatography plate and then the Capto eluent was collected by washing away impurities and gradient elution.

[0016] Step 4, Biomimetic Base Layer Analysis

[0017] After diluting the Capto eluent with ultrapure water to reduce conductivity, the eluent was subjected to biomimetic substrate chromatography. The eluent was collected in flow-through mode, concentrated by ultrafiltration, and then sterilized and stored.

[0018] Furthermore, in step one, the acid precipitation process includes: adding 45-55% acetic acid to the ultrafiltration concentrate, adjusting the pH, stirring and stabilizing the pH, adjusting the temperature, and allowing it to stand. As an example, preferably, 45-55% acetic acid is added to the ultrafiltration concentrate to adjust the pH to 4-5, stirring at 25-35 Hz for at least 5 minutes to stabilize the pH, adjusting the temperature to 3-8°C, and allowing it to stand for 30-120 minutes. This removes most of the positively charged proteins, reducing the burden on subsequent chromatography.

[0019] A tangential flow ultrafiltration system (4m) was adopted. 2 The acid precipitation centrifuge solution was replaced with phosphate buffer (PBS) to match the chromatography system.

[0020] Furthermore, in step two, the anion exchange chromatography packing material is the high-resolution weak anion adsorption packing material DEAE Bestrose HP. Column efficiency: ≥2000 plates / m (acetone test method); MCC300 type is preferred.

[0021] As an example of a flow-through mode program:

[0022] Equilibration: Equilibrate the anion exchange chromatography column with 0.1-0.3M PBS (pH=7.0-7.4) at a flow rate of 120-180 cm / h and a volume of 5 CV.

[0023] Sample loading: Collect the pretreated crude extract at a flow rate of 120-180 cm / h, a volume of 15 CV, and a UV206 concentration of ≥300 mAU.

[0024] Washing: Wash the anion exchange chromatography column with 0.1-0.3M PBS (pH=7.0-7.4) at a flow rate of 120-180cm / h and a volume of 3CV.

[0025] Regeneration: 1-3M NaCl solution, flow rate 100cm / h, volume 2CV, UV280 to zero.

[0026] Collection interval: from UV206 ≥ 300 mAU to signal drop to baseline (polysaccharide recovery ≥ 95%).

[0027] Furthermore, in step two, the ultrafiltration buffer change is mainly to connect with the Capto elution in the subsequent composite chromatography, and the target buffer is 15-25mM Tris-HCl (pH=7.0-7.4).

[0028] Furthermore, in step three, the packing material selected for composite chromatography is high-resolution Capto QImpRes.

[0029] Furthermore, in step four, the packing material for the biomimetic ligand-based chromatography is selected from biomimetic ligand packing materials. Serum-type ligands 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 20, 22F, and 33F are shown in SEQ ID NO.1; serum-type ligand 6B is shown in SEQ ID NO.2; serum-type ligand 19F is shown in SEQ ID NO.3; and serum-type ligand 23F is shown in SEQ ID NO.4.

[0030] Specifically:

[0031] SEQ ID NO.1: H2N-Gly-Arg-Trp-Pro-Ser-Lys-Thr-Tyr-COOH;

[0032] SEQ ID NO.2: H2N-Gly-Cit-Trp-Ser-Ser-Tyr-COOH;

[0033] SEQ ID NO.3: H2N-Arg-Trp-His-Gly-Ser-Tyr-NH2;

[0034] SEQ ID NO.4: H2N-Arg-Trp-His-Gly-Ser-Tyr-COOH+ZnCl2 (0.1mM).

[0035] Because serum type 6B contains phosphoric acid choline (strongly negatively charged), Arg is reduced and citrulline (Cit) is introduced to reduce its charge; serum type 19F has a high O-acetyl group (large steric hindrance), so it is shortened to a 6-peptide and C-terminal amidation is used for blocking; serum type 23F has ribitol phosphate (weakly negatively charged) with His chelation of Zn. 2+ Enhance integration.

[0036] The specific preparation method of the packing material is as follows: Cleaning: Take 100 mL of precipitated gel, rinse 3 times with ultrapure water (5 column volumes / time), and vacuum filter until the water content is ≤5%. Immerse in 20% ethanol and store at 4℃ for later use.

[0037] Epoxy group activation: Take 100 mL of precipitated gel and add 200 mL of 1,4-butanediol diglycidyl ether (0.2 M NaOH dissolved in the activator). Shake at 25 °C (150 rpm) for 4 hours. After the reaction, wash with 5 CV each of ultrapure water, 0.1 M acetic acid (pH 4.0) and 20% ethanol to obtain activated gel.

[0038] Peptide solution: Serum-type ligand 2 mg / mL, dissolved in 0.1 M carbonate buffer, pH 8.5;

[0039] The activated gel was dried, and a polypeptide solution with a sedimentation volume twice that of the gel was added. Nitrogen gas was purged to remove oxygen, and the mixture was sealed and shaken at 40°C to maintain the pH at 8.5±0.2. Then, 1M ethanolamine (pH 9.0) was added, and the mixture was shaken at 25°C for 2 hours. The mixture was then washed sequentially with 6M urea (5CV), 0.1M acetic acid (5CV), and 20% ethanol (5CV) to obtain the biomimetic ligand filler.

[0040] Furthermore, in step four, the flow-through mode collection includes equilibration, sample loading, and elution collection.

[0041] As an example, the preferred option is:

[0042] Equilibration: Equilibrate the chromatography column with 5CV equilibration buffer and 20mM Tris-HCl. If it is serum type 23F, 0.1mM ZnCl2 solution should also be added.

[0043] Sample loading: Load the diluted sample solution (sample loading amount ≤ 15 mg polysaccharide / mL filler);

[0044] Flow-through mode collection: UV260 is monitored throughout the entire process, and collection is performed when the signal is <50mAU;

[0045] regeneration:

[0046] 0.5M NaCl + 5mM EDTA (dissociates nucleic acids), 0.1M NaOH (CIP).

[0047] The present invention has the following advantages:

[0048] The purification process of this invention first uses acid precipitation to remove 30% of the protein and nucleic acid. Then, anion exchange chromatography is used for crude extraction to remove more than 80% of the protein and nucleic acid. Anion exchange chromatography can handle large volumes of feed solution. Next, composite chromatography is used for purification to remove residual protein, nucleic acid and endotoxin, and the polysaccharide is further concentrated at high loading. Finally, biomimetic substrate chromatography is used for deep impurity removal and targeted removal of trace nucleic acid. The final result is a single polysaccharide recovery rate of more than 90%, a protein content of less than 0.1%, a nucleic acid content of less than 0.1%, an endotoxin content of less than 0.1 EU / mL, and a removal rate of more than 99%.

[0049] The purification process of this invention adopts a flow-through mode in both anion exchange chromatography and biomimetic substrate chromatography, which has almost no adsorption on single polysaccharides, thus further improving the polysaccharide recovery rate. In the composite chromatography stage, the elution salt concentration is precisely controlled to avoid polysaccharide retention, further improving the polysaccharide recovery rate. Attached Figure Description

[0050] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0051] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0052] Figure 1 This is a chromatogram of polysaccharide purity provided in Embodiment 1 of the present invention;

[0053] Figure 2 This is a chromatogram of polysaccharide purity provided in Example 2 of the present invention. Detailed Implementation

[0054] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] Solid culture medium (blood agar): Guangdong Huankai Microbial Technology Co., Ltd., 024070;

[0056] Pneumococcus: ATCC 49619.

[0057] Example 1

[0058] This embodiment provides a purification process for pneumococcal monovalent polysaccharide 10A:

[0059] Step 1, Pre-processing

[0060] Pneumococcus was cultured on solid culture medium (blood agar) at 37°C for 18-24 hours; then in a three-stage fermenter (50L→500L→5000L) for 12 hours with dissolved oxygen at 30% and pH=7.0; treated with 0.5% formaldehyde in a fire extinguishing canister at 37°C for 2 hours; centrifuged at 8000×g at 4°C for 30 minutes; and concentrated to a polysaccharide concentration of 15-20 mg / mL using a tangential flow ultrafiltration system with a 10 kDa membrane and a transmembrane pressure of 1.0-1.5 bar to obtain the ultrafiltration concentrate.

[0061] Acid precipitation for impurity removal: Transfer the ultrafiltration concentrate to an acid precipitation tank and turn on the stirrer (30Hz); slowly add 50% acetic acid through the pre-reserved opening at the top of the tank to adjust the pH to 4.5±0.2; continue stirring for 5 min, then turn off the stirrer; turn on the tank temperature control to 5℃ and let it stand for 90 min; centrifuge: 6000×g, 4℃, 20 min, collect the supernatant to obtain the acid precipitation impurity removal solution; this part can achieve a protein removal rate ≥85% and a nucleic acid removal rate ≥50%.

[0062] Ultrafiltration fluid replacement: A tangential flow ultrafiltration system (4m) is used. 2 Membrane pack (5 kDa), transmembrane pressure (TMP): 0.5-1.0 bar, dialysis buffer: 0.2 M PBS (pH 7.2). Replace the acid precipitation centrifugation buffer with phosphate buffer (PBS) to match the chromatography system. Endpoint criteria: conductivity 18 ± 2 mS / cm, pH 7.2 ± 0.1 (consistent with the DEAE equilibration system).

[0063] Step 2, Anion Chromatography

[0064] Packing material: DEAE Bestrose HP;

[0065] Column specifications: MCC300 type (diameter 30cm, column height 20cm);

[0066] Column loading efficiency: ≥2000 plates / m (acetone test method).

[0067] Equilibration: The anion exchange chromatography column was equilibrated with 0.2M PBS (pH=7.2) at a flow rate of 150cm / h and a volume of 15CV.

[0068] Sample loading: Collect the pretreated crude extract at a flow rate of 150 cm / h, a volume of 15 CV, and a UV206 concentration of ≥300 mAU.

[0069] Washing: Wash the anion exchange chromatography column with 0.2M PBS (pH=72) at a flow rate of 150cm / h and a volume of 3CV.

[0070] Regeneration: 2M NaCl solution, flow rate 100cm / h, volume 2CV, UV280 reset to zero.

[0071] Collection interval: from UV206 ≥ 300 mAU to signal drop to baseline, DEAE-treated solution was obtained; polysaccharide recovery rate was 98.7%, protein 2.3%, and nucleic acid 1%.

[0072] Ultrafiltration buffer change: Target buffer: 20 mM Tris-HCl (pH 7.4); Endpoint conductivity: ≤5 mS / cm; Polysaccharide concentration: 20 ± 2 mg / mL.

[0073] Step 3, Composite Chromatography

[0074] Packing material: Capto Q ImpRes (high resolution); Column specifications: XK 50 / 100 (5cm diameter, 10cm column height);

[0075] Equilibration: The composite chromatography column was equilibrated with 20 mM Tris-HCl (pH 7.4) at a flow rate of 60 cm / h and a volume of 5 CV.

[0076] Sample loading: The DEAE treatment solution was loaded at a flow rate of 30 cm / h and the volume was increased to 60 mg polysaccharide / mL of filler. Baseline was set at UV280.

[0077] Washing: Use 20 mM Tris-HCl, flow rate 60 cm / h, volume 2 CV, UV260 < 50 mAU.

[0078] Gradient elution: 0→0.3M NaCl (linear slope of 0.03M / CV), flow rate 30cm / h, volume 10CV, collect the 0.15-0.25M NaCl range to obtain Capto eluent, polysaccharide recovery rate 98%, protein 1.28%, nucleic acid 0.1%, endotoxin 1EU / mL.

[0079] Regeneration: 1M NaCl solution, flow rate 100cm / h, volume 2CV, UV280 reset to zero, 0.1M NaOH (CIP).

[0080] Dilution: Dilute the Capto Q eluent 3 times with ultrapure water to make the conductivity ≤3mS / cm to obtain the diluted solution.

[0081] Step 4, Biomimetic Base Layer Analysis

[0082] The ligand is shown in SEQ ID NO.1;

[0083] Washing: Take 100 mL of the settled gel and wash it 3 times with ultrapure water (5 column volumes / wash). Vacuum filter until the water content is ≤5%. Immerse it in 20% ethanol and store at 4°C for later use.

[0084] Epoxy group activation: Take 100 mL of precipitated gel and add 200 mL of 1,4-butanediol diglycidyl ether (0.2 M NaOH dissolved in the activator). Shake at 25 °C (150 rpm) for 4 hours. After the reaction, wash with 5 CV each of ultrapure water, 0.1 M acetic acid (pH 4.0) and 20% ethanol to obtain activated gel.

[0085] Peptide solution: 2 mg / mL serum-type 10A ligand dissolved in 0.1 M carbonate buffer, pH 8.5;

[0086] The activated gel was dried, and a polypeptide solution with a sedimentation volume twice that of the gel was added. Nitrogen gas was purged to remove oxygen, and the mixture was sealed and shaken at 40°C to maintain the pH at 8.5±0.2. Then, 1M ethanolamine (pH 9.0) was added, and the mixture was shaken at 25°C for 2 hours. The mixture was then washed sequentially with 6M urea (5CV), 0.1M acetic acid (5CV), and 20% ethanol (5CV) to obtain the biomimetic ligand filler.

[0087] Equilibration: The biomimetic substrate chromatography column was equilibrated with 20 mM Tris-HCl (pH 7.4) at a flow rate of 60 cm / h and a volume of 5 CV.

[0088] Sample loading: The diluent was loaded at a flow rate of 10 cm / h, with a loading volume ≤ 15 mg polysaccharide / mL packing material. UV260 was monitored throughout the process, and the sample was collected when the signal was < 50 mAU to obtain purified monopolysaccharide solution. The polysaccharide recovery rate was 97.2%, protein 0.018%, nucleic acid 0.01%, and endotoxin 0.05 EU / mL. The polysaccharide purity chromatogram is shown below. Figure 1 As shown, the main peak is regular and symmetrical, with no obvious impurity peaks, proving that the polysaccharide has a high purity.

[0089] Regeneration: 0.5M NaCl + 5mM EDTA (dissociates nucleic acids), 0.1M NaOH (CIP).

[0090] Post-processing:

[0091] Ultrafiltration concentration: Concentrate to a polysaccharide concentration of ≥20mg / mL using a 10kDa membrane; Sterilization filtration: Filter using a 0.22μm PES membrane; Storage: Temporarily store at 2-8℃ or freeze-dry.

[0092] Example 2

[0093] This embodiment provides a purification process for pneumococcal monovalent polysaccharide 6B:

[0094] This embodiment is identical to Example 1 except for the polypeptide solution and serum-type 6B ligand.

[0095] The polysaccharide recovery rate, protein content, and nucleic acid content at each step are as follows:

[0096] DEAE-treated solution; polysaccharide recovery rate 99.1%, protein 1.8%, nucleic acid 1.1%;

[0097] Capto eluent: Polysaccharide recovery rate 98.2%, protein 1.22%, nucleic acid 0.12%, endotoxin 1.1 EU / mL;

[0098] The single polysaccharide solution showed a polysaccharide recovery rate of 98.5%, protein 0.012%, nucleic acid 0.0%, and endotoxin 0.01 EU / mL. The polysaccharide purity chromatogram is shown below. Figure 2 As shown, the main peak is regular and symmetrical, with no obvious impurity peaks, proving that the polysaccharide has a high purity.

[0099] Example 3

[0100] This embodiment provides a purification process for pneumococcal monovalent polysaccharide 19F:

[0101] This embodiment is identical to Example 1 except for the polypeptide solution and serum-type 19F ligand.

[0102] The polysaccharide recovery rate, protein content, and nucleic acid content at each step are as follows:

[0103] DEAE-treated solution; polysaccharide recovery rate 97.1%, protein 1.2%, nucleic acid 0.9%;

[0104] Capto eluent: Polysaccharide recovery rate 98.7%, protein 0.82%, nucleic acid 0.11%, endotoxin 1.1 EU / mL;

[0105] The single polysaccharide solution showed a polysaccharide recovery rate of 98.1%, a protein recovery rate of 0.011%, a nucleic acid recovery rate of 0.0%, and an endotoxin recovery rate of 0.02 EU / mL.

[0106] Example 4

[0107] This embodiment provides a purification process for pneumococcal monovalent polysaccharide 23F:

[0108] This embodiment is identical to Example 1 except that the polypeptide solution and serum-type 23F ligand are the same, and 0.1 mM ZnCl2 needs to be added to the equilibration solution in step four.

[0109] The polysaccharide recovery rate, protein content, and nucleic acid content at each step are as follows:

[0110] DEAE-treated solution; polysaccharide recovery rate 96.1%, protein 1.5%, nucleic acid 1.3%;

[0111] Capto eluent: Polysaccharide recovery rate 95.1%, protein 1.1%, nucleic acid 0.21%, endotoxin 1.2 EU / mL;

[0112] The single polysaccharide solution showed a polysaccharide recovery rate of 98.7%, a protein recovery rate of 0.015%, a nucleic acid recovery rate of 0.01%, and an endotoxin recovery rate of 0.01 EU / mL.

[0113] Comparative Example 1

[0114] This comparative example provides a purification process for pneumococcal monovalent polysaccharide 10A:

[0115] In this comparative example, biomimetic substrate chromatography was replaced with Hydroxyapatite chromatography, but otherwise it was completely consistent with Example 1.

[0116] The purified monopolysaccharide solution obtained had a polysaccharide recovery rate of 75.1%, a protein recovery rate of 0.55%, a nucleic acid recovery rate of 0.03%, and an endotoxin recovery rate of 1.1 EU / mL.

[0117] In Hydroxyapatite chromatography, calcium ions bind to the phosphate groups of polysaccharides, leading to irreversible adsorption loss of the polysaccharides.

[0118] Hydroxyapatite has a weak affinity for nucleic acids and is not very effective at removing them.

[0119] Hydroxyapatite relies on calcium and phosphorus adsorption and has a low scavenging rate for lipopolysaccharide (LPS).

[0120] Comparative Example 2

[0121] This comparative example provides a purification process for pneumococcal monovalent polysaccharide 10A:

[0122] In this comparative example, the gradient elution in the composite chromatography was replaced with constant elution with 0.2M NaCl, and everything else was exactly the same as in Example 1.

[0123] The purified monopolysaccharide solution obtained had a polysaccharide recovery rate of 85.3%, a protein recovery rate of 0.82%, a nucleic acid recovery rate of 0.25%, and an endotoxin recovery rate of 0.9 EU / mL.

[0124] Constant current elution (0.2M NaCl) resulted in the failure to elute weakly bound polysaccharides, leading to a loss of 12% of the polysaccharides.

[0125] During constant current elution, nucleic acids, endotoxins, and polysaccharides are co-eluted, resulting in a low elution rate.

[0126] Comparative Example 3

[0127] This comparative example provides a purification process for pneumococcal monovalent polysaccharide 10A:

[0128] In this comparative example, the flow rate of the diluent in the biomimetic substrate analysis was adjusted to 20 cm / h, while everything else was completely consistent with Example 1.

[0129] The purified monopolysaccharide solution obtained had a polysaccharide recovery rate of 90.5%, a protein recovery rate of 0.035%, a nucleic acid recovery rate of 0.12%, and an endotoxin recovery rate of 0.08 EU / mL.

[0130] Doubling the flow rate in biomimetic chromatography leads to insufficient nucleic acid adsorption kinetics, resulting in increased penetration. To ensure purity, the collection was terminated early, resulting in a loss of 6.7% of polysaccharides.

[0131] High flow rates (20 cm / h) resulted in insufficient nucleic acid-ligand contact time, leading to increased penetration. Endotoxin clearance was less affected by flow rate, thus remaining close to the levels observed in Example 1.

[0132] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A purification process for a monovalent polysaccharide from pneumococcus, characterized in that, The process includes: Step 1, Pre-processing The pneumococcal monopolysaccharide was centrifuged to remove bacterial cells, and the solution was concentrated using ultrafiltration. Acid precipitation was used to initially remove impurities, including some proteins and nucleic acids. The acid-precipitated centrifuged solution was then pretreated with an ultrafiltration system and added to a chromatographic equilibrium system PBS to obtain a pretreated crude extract. Step 2, Anion Chromatography The pretreated crude extract was passed through an anion exchange chromatography column, and the feed solution was collected in flow-through mode to obtain DEAE flow-through solution. The solution was then switched to a low conductivity Tris buffer system for ultrafiltration to obtain DEAE treated solution. The anion chromatography packing material is a high-resolution weak anion adsorption packing material, DEAE Bestrose HP; The procedure for the flow-through mode is as follows: Equilibration: Equilibrate the anion exchange chromatography column with 0.1-0.3M PBS, pH=7.0-7.4, at a flow rate of 120-180cm / h and a volume of 5CV; Sample loading: Collect the pretreated crude extract at a flow rate of 120-180 cm / h, a volume of 15 CV, and a UV206 concentration of ≥300 mAU; Washing: Wash the anion exchange chromatography column with 0.1-0.3M PBS, pH=7.0-7.4, at a flow rate of 120-180cm / h and a volume of 3CV; Regeneration: 1-3M NaCl solution, flow rate 100cm / h, volume 2CV, UV280 reset to zero; Step 3, Composite Chromatography After loading the DEAE-treated solution onto a composite chromatography column, the Capto eluent was collected by washing away impurities and gradient elution. The specific steps are as follows: Packing material: Capto Q ImpRes; Equilibration: The composite chromatography column was equilibrated with 20 mM Tris-HCl at pH 7.4 at a flow rate of 60 cm / h and a volume of 5 CV. Sample loading: The DEAE treatment solution was loaded at a flow rate of 30 cm / h and the volume was increased to 60 mg polysaccharide / mL of filler material. Baseline was set at UV280. Washing: Use 20 mM Tris-HCl, flow rate 60 cm / h, volume 2 CV, UV260 < 50 mAU; Gradient elution: 0→0.3 M NaCl, linear slope of 0.03 M / CV, flow rate of 30 cm / h, volume of 10 CV, collect the 0.15-0.25 M NaCl range to obtain Capto eluent; Regeneration: 1M NaCl solution, flow rate 100cm / h, volume 2CV, UV280 reset to zero, 0.1M NaOH; Dilution: Dilute the Capto Q eluent 3 times with ultrapure water to make the conductivity ≤3 mS / cm to obtain the diluted solution; Step 4, Biomimetic Base Layer Analysis After diluting the Capto eluent with ultrapure water to reduce conductivity, the eluent was subjected to biomimetic substrate chromatography. The eluent was collected in flow-through mode, concentrated by ultrafiltration, and then sterilized and stored. The flow-through mode collection includes equilibration, loading, and elution collection; The equilibration process involves using a 5CV equilibration buffer and a 20mM Tris-HCl column for column equilibration; for serum type 23F, 0.1 mM ZnCl2 solution is added to the equilibration buffer; the loading speed is 10 cm / h. The biomimetic ligand-based chromatography packing material is selected from biomimetic ligand packing materials, which are prepared by combining serum-type ligands with sedimentation gels. The sedimentation gel is first activated by epoxy groups to obtain an activated gel, which is then combined with serum-type ligands to prepare the biomimetic ligand packing material. The specific serum-type ligands are: serum-type 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 20, 22F, 33F ligands such as H2. The N-Gly-Arg-Trp-Pro-Ser-Lys-Thr-Tyr-COOH ligand is shown in H2N-Gly-Cit-Trp-Ser-Ser-Tyr-COOH; the serotype 19F ligand is shown in H2N-Arg-Trp-His-Gly-Ser-Tyr-NH2; and the serotype 23F ligand is shown in H2N-Arg-Trp-His-Gly-Ser-Tyr-COOH. The specific preparation method of the filler is as follows: Washing: Take 100 mL of the settled gel, rinse 3 times with ultrapure water (5 column volumes per rinse), and vacuum filter until the water content is ≤5%; immerse in 20% ethanol and store at 4℃ for later use; Epoxy group activation: 100 mL of precipitated gel was added to 200 mL of 1,4-butanediol diglycidyl ether. The activator contained 0.2 M NaOH. The mixture was shaken at 150 rpm at 25 °C for 4 hours. After the reaction, the gel was washed with 5 CV each of ultrapure water, 0.1 M acetic acid, pH=4.0 and 20% ethanol to obtain the activated gel. Peptide solution: Serum-type ligand 2 mg / mL, dissolved in 0.1 M carbonate buffer, pH 8.5; The activated gel was dried, and a polypeptide solution with a sedimentation volume twice that of the gel was added. Nitrogen gas was purged to remove oxygen, and the mixture was sealed and shaken at 40°C to maintain the pH at 8.5±0.

2. Then, 1M ethanolamine was added to bring the pH to 9.0, and the mixture was shaken at 25°C for 2 hours. The mixture was then washed sequentially with 6M urea 5CV, 0.1M acetic acid 5CV, and 20% ethanol 5CV to obtain the biomimetic ligand filler.

2. The purification process for a pneumococcal monovalent polysaccharide according to claim 1, characterized in that, In step one, the acid precipitation process includes: adding 45-55% acetic acid to the ultrafiltration concentrate, adjusting the pH, stirring and stabilizing the pH, adjusting the temperature, and letting it stand.

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