Purification process of pneumococcus monovalent polysaccharide

Abstract

The invention discloses a purification process of pneumococcus monovalent polysaccharide, and particularly relates to the technical field of biological medicine. The process comprises pretreatment, anion chromatography, composite chromatography and bionic ligand chromatography. According to the purification technology, 30% of protein and nucleic acid are removed through an acid precipitation technology, then crude extraction is conducted through anion chromatography, more than 80% of protein and nucleic acid are removed, and large-volume feed liquid can be treated through anion chromatography crude extraction; performing compound chromatography purification to remove residual protein, nucleic acid and endotoxin, and further concentrating polysaccharide at high load; and finally, trace nucleic acid is cleared in a targeted manner through bionic ligand chromatography, deep impurity removal and removal, the recovery rate of the single polysaccharide is 90% or above, the protein content is lower than 0.1%, the nucleic acid content is lower than 0.1%, the endotoxin content is lower than 0.1 EU / mL, and the removal rate reaches 99% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a purification process of pneumococcal monovalent polysaccharide. Background Art

[0002] Streptococcus pneumoniae is a Gram-positive bacterium with over 90 serotypes, most of which are coated with a capsular polysaccharide. Capsular polysaccharides can evade recognition by the immune system and prevent phagocytosis by immune cells, allowing the bacteria to multiply in the body and cause disease. Years of research and clinical trials have demonstrated that the capsular polysaccharide of Streptococcus pneumoniae can be used as a vaccine to induce specific antibodies and provide excellent immune protection against the corresponding Streptococcus pneumoniae. The capsular polysaccharides of different serotypes of Streptococcus pneumoniae vary in composition and structure, so a combination of polysaccharides from different serotypes of Streptococcus pneumoniae is often required as a vaccine to prevent the pathogenicity of the main Streptococcus pneumoniae strain.

[0003] During the fermentation process, Streptococcus pneumoniae, used to prepare polysaccharide vaccines, produces not only capsular polysaccharides but also a large number of metabolites. To achieve the required capsular polysaccharide purity for vaccine preparation, the fermentation broth typically undergoes multiple purification steps to remove impurities such as proteins, nucleic acids, and endotoxins from the crude polysaccharide product, thereby preventing potential side effects associated with subsequent vaccination.

[0004] The commonly used ethanol precipitation method is generally effective in removing protein contaminants from polysaccharides, but it struggles to achieve the purity required for injectable vaccines. Furthermore, the use of ethanol presents the following challenges: 1) Its use requires fire-resistant facilities, which are expensive to design; 2) The required volume of ethanol is enormous, requiring approximately 4-6 liters per liter of processed material, and wastewater disposal is extremely difficult; 3) Reconstitution of the treated polysaccharide is difficult, requiring freeze-drying and other steps.

[0005] Chinese patent CN112646050A discloses a process for purifying pneumococcal polysaccharides using ion exchange chromatography. This patent employs an ion exchange chromatography method for purifying pneumococcal polysaccharides. However, this method has a low sample loading capacity. As the sample loading capacity increases, the protein and nucleic acid content increases significantly, resulting in a low removal rate. Although the low sample loading capacity can ensure the removal rate, the processing capacity is insufficient and the efficiency is too low.

[0006] Chinese patent CN107043431A, a method for purifying bacterial capsular polysaccharides, uses composite ion exchange chromatography and hydroxyphosphate gray salt chromatography for chromatography to obtain a purified capsular polysaccharide solution. However, using composite chromatography alone, the recovery rate of the obtained product is only 60 to 80%, the protein content is greater than 0.5%, and the removal rate is low. In addition, this patent still faces the problem of small sample volume. Summary of the Invention

[0007] To this end, the present invention provides a purification process for pneumococcal monovalent polysaccharide to solve the problems in the prior art.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] According to the present invention, a purification process for a monovalent pneumococcal polysaccharide is provided, the process comprising:

[0010] Step 1: Pre-treatment

[0011] After the pneumococci are activated, expanded, and fermented in a fermenter, the polysaccharide is harvested by inactivation, the bacteria are removed by centrifugation, the feed solution is concentrated by ultrafiltration, impurities including some proteins and nucleic acids are preliminarily removed by acid precipitation, and the acid precipitation centrifuge is pretreated by an ultrafiltration system into a chromatographic equilibrium system PBS to obtain a pretreated crude extract;

[0012] Step 2, anion chromatography

[0013] The crude extract after pretreatment was passed through an anion chromatography column, and the feed liquid was collected in flow-through mode to obtain DEAE flow-through liquid, which was switched to a low-conductivity Tris buffer system for ultrafiltration to obtain a DEAE treated liquid;

[0014] Step 3: Composite chromatography

[0015] The initial concentrated polysaccharide solution was loaded onto composite chromatography, and then washed and gradient eluted to obtain the Capto eluate.

[0016] Step 4: Bionic matching analysis

[0017] The Capto eluate was diluted with ultrapure water to reduce the conductivity, and then a biomimetic fluoropolymer layer was applied for separation. The eluate was collected in flow-through mode, concentrated by ultrafiltration, and sterilized and filtered for storage.

[0018] Furthermore, in step 1, the acid precipitation process includes adding 45-55% acetic acid to the ultrafiltration concentrate, adjusting the pH, stirring to stabilize the pH, adjusting the temperature, and allowing the solution 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 the solution to stand for 30-120 minutes. This removes most of the positively charged proteins, reducing the burden on subsequent chromatography.

[0019] Using tangential flow ultrafiltration system (4m 2 membrane package), replace the acid precipitation centrifuge liquid with phosphate buffered saline (PBS) and match the chromatography system.

[0020] Furthermore, in step 2, the anion chromatography filler is a high-resolution weak anion adsorption filler DEAE Bestrose HP, with a column efficiency of ≥2000 plates / m (acetone test method), preferably MCC300.

[0021] As an example, the flow-through mode program is:

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

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

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

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

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

[0027] Furthermore, in step 2, the ultrafiltration buffer change is mainly for connecting to the Capto elution in the subsequent composite chromatography, and the target buffer is 15-25 mM Tris-HCl (pH = 7.0-7.4).

[0028] Furthermore, in step 3, the filler selected for composite chromatography is high-resolution Capto QImpRes.

[0029] Furthermore, in the step 4, the filler for the bionic ligand analysis is a bionic ligand filler, and the serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 20, 22F, and 33F ligands are shown in SEQ ID NO.1, the serotype 6B ligand is shown in SEQ ID NO.2; the serotype 19F ligand is shown in SEQ ID NO.3; and the serotype 23F ligand 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 serotype 6B contains phosphorylcholine (strong negative charge), Arg is reduced and citrulline (Cit) is introduced to reduce the charge; serotype 19F, with high O-acetyl groups (large steric hindrance), is shortened to 6 peptides and the C-terminus is amidated and blocked; serotype 23F, with ribitol phosphate (weakly negative charge), adds His to chelate Zn 2+ Enhance bonding.

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

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

[0038] Peptide solution: 2 mg / mL of serotype ligand in 0.1 M carbonate buffer, pH 8.5;

[0039] The activated gel was drained, and a polypeptide solution twice the volume of the gel sedimentation volume was added. Nitrogen was passed through and oxygen was removed. After sealing, the reaction was shaken at 40°C, maintaining the pH at 8.5±0.2. 1M ethanolamine (pH 9.0) was added and shaken at 25°C for 2 hours. The gel was washed with 6M urea (5CV), 0.1M acetic acid (5CV) and 20% ethanol (5CV) in sequence to obtain the biomimetic ligand filler.

[0040] Furthermore, in step 4, the flow-through mode collection includes equilibration, sample loading, and elution collection.

[0041] As an example, it is preferred that:

[0042] Equilibration: Equilibrate the column with 5CV of 20mM Tris-HCl equilibration buffer. If the serotype is 23F, add 0.1mM ZnCl2 solution.

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

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

[0045] regeneration:

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

[0047] The present invention has the following advantages:

[0048] The purification process of the present invention first uses an acid precipitation process to remove 30% of proteins and nucleic acids, then uses anion chromatography for crude extraction to remove more than 80% of proteins and nucleic acids, and the anion chromatography crude extraction can process a large volume of feed liquid; then uses composite chromatography for purification to remove residual proteins, nucleic acids and endotoxins, and further concentrates polysaccharides at a high load; finally, uses biomimetic ligand chromatography for deep impurity removal and targeted removal of trace nucleic acids, ultimately obtaining 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 the present invention adopts a flow-through mode in both anion chromatography and biomimetic ligand chromatography, which has almost no adsorption to single polysaccharides and can further improve the recovery rate of polysaccharides. In the composite chromatography stage, the elution salt concentration is accurately controlled to avoid the retention of polysaccharides and further improve the recovery rate of polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0051] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0052] Figure 1 A polysaccharide purity chromatogram provided in Example 1 of the present invention;

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

[0054] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

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

[0056] Pneumococcus: ATCC 49619.

[0057] Example 1

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

[0059] Step 1: Pre-treatment

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

[0061] Acid precipitation and impurity removal: transfer the ultrafiltration concentrate to an acid precipitation tank and start stirring (30 Hz); slowly add 50% acetic acid from the reserved port on the tank top to adjust the pH to 4.5±0.2; continue stirring for 5 minutes, then turn off stirring; turn on the tank temperature control to 5°C and let it stand for 90 minutes; centrifuge: 6000×g, 4°C, 20 minutes, collect the supernatant to obtain the acid precipitation and impurity removal liquid; this part can achieve a protein removal rate of ≥85% and a nucleic acid removal rate of ≥50%.

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

[0063] Step 2, anion chromatography

[0064] Filler: DEAE Bestrose HP;

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

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

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

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

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

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

[0071] Collection interval: UV206 ≥ 300 mAU until the signal drops to the baseline, obtaining the DEAE-treated solution; the recovery rates of polysaccharides are 98.7%, proteins 2.3%, and nucleic acids 1%.

[0072] Ultrafiltration solution exchange: 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 (5 cm diameter, 10 cm 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 flowed at a rate of 30 cm / h and the volume was adjusted to 60 mg polysaccharide / mL filler, with UV280 baseline.

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

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

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

[0080] Dilution: Dilute the Capto Q eluate 3-fold with ultrapure water to obtain a dilution solution with a conductivity of ≤3 mS / cm.

[0081] Step 4: Bionic matching analysis

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

[0083] Washing: Take 100 mL of the precipitated gel, rinse with ultrapure water three times (5 column volumes / time), vacuum filter until the water content is ≤5%, immerse in 20% ethanol, and store at 4°C until use.

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

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

[0086] The activated gel was drained, and a polypeptide solution twice the volume of the gel sedimentation volume was added. Nitrogen was passed through and oxygen was removed. After sealing, the reaction was shaken at 40°C, maintaining the pH at 8.5±0.2. 1M ethanolamine (pH 9.0) was added and shaken at 25°C for 2 hours. The gel was washed with 6M urea (5CV), 0.1M acetic acid (5CV) and 20% ethanol (5CV) in sequence to obtain the biomimetic ligand filler.

[0087] Equilibration: The biomimetic ligand 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 flow rate was 10 cm / h, the sample load was ≤15 mg polysaccharide / mL filler, UV260 was monitored throughout the process, and the sample was collected when the signal was <50 mAU. The purified single polysaccharide solution was obtained with a polysaccharide recovery rate of 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 miscellaneous peaks, proving that the polysaccharide has a high purity.

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

[0090] Post-processing:

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

[0092] Example 2

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

[0094] This example only uses the polypeptide solution: serotype 6B ligand, and the rest is exactly the same as in Example 1.

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

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

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

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

[0099] Example 3

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

[0101] This example only uses the polypeptide solution: serotype 19F ligand, and the rest is exactly the same as in Example 1.

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

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

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

[0105] Single polysaccharide solution, polysaccharide recovery rate 98.1%, protein 0.011%, nucleic acid 0.0%, endotoxin 0.02EU / mL.

[0106] Example 4

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

[0108] In this example, only the polypeptide solution: serotype 23F ligand, 0.1 mM ZnCl2 needs to be added to the equilibrium solution in step 4, and the rest is exactly the same as in Example 1.

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

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

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

[0112] Single polysaccharide solution, polysaccharide recovery rate 98.7%, protein 0.015%, nucleic acid 0.01%, endotoxin 0.01EU / mL.

[0113] Comparative Example 1

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

[0115] In this comparative example, the biomimetic ligand chromatography was replaced by Hydroxyapatite chromatography, and the other procedures were the same as those in Example 1.

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

[0117] Calcium ions bind to the phosphate groups of polysaccharides during hydroxyapatite chromatography, resulting in irreversible adsorption loss of polysaccharides.

[0118] Hydroxyapatite has weak adsorption capacity for nucleic acids and is not very effective in removing them.

[0119] Hydroxyapatite relies on calcium and phosphate adsorption and has a low clearance 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 a constant elution with 0.2 M NaCl, and the other steps were the same as those in Example 1.

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

[0124] Constant flow elution (0.2 M NaCl) resulted in the non-elution of weakly bound polysaccharides, resulting in a loss of 12% of polysaccharides.

[0125] During constant flow elution, nucleic acids, endotoxins and polysaccharides are co-eluted, and the elution rate is low.

[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 ligand analysis was adjusted to 20 cm / h, and the other procedures were completely consistent with those in Example 1.

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

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

[0131] The high flow rate (20 cm / h) resulted in insufficient nucleic acid-ligand contact time and increased penetration. Endotoxin clearance was less affected by flow rate and was close to the level of Example 1.

[0132] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A process for purifying pneumococcal monovalent polysaccharide, characterized in that: The process comprises: Step 1: Pre-treatment The single polysaccharide of pneumococcus is centrifuged to remove the bacteria, and the feed liquid is concentrated by ultrafiltration concentration process. The impurities including some proteins and some nucleic acids are preliminarily removed by acid precipitation process. The acid precipitation centrifuge liquid is pretreated by ultrafiltration system and transferred to PBS, a chromatography equilibrium system, to obtain a pretreated crude extract. Step 2, anion chromatography The crude extract after pretreatment was passed through an anion chromatography column, and the feed liquid was collected in flow-through mode to obtain DEAE flow-through liquid, which was switched to a low-conductivity Tris buffer system for ultrafiltration to obtain a DEAE treated liquid; Step 3: Composite chromatography The initial concentrated polysaccharide solution was loaded onto composite chromatography, and then washed and gradient eluted to obtain the Capto eluate. Step 4: Bionic matching analysis The Capto eluate was diluted with ultrapure water to reduce the conductivity, and then a biomimetic fluoropolymer layer was applied for separation. The eluate was collected in flow-through mode, concentrated by ultrafiltration, and sterilized and filtered for storage.

2. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 1, 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 the solution to stand.

3. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 2, the anion chromatography filler is a high-resolution weak anion adsorption filler, DEAE Bestrose HP.

4. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 2, the flow-through mode includes equilibration, sample loading, washing and collection.

5. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In step 3, the filler selected for composite chromatography is high-resolution Capto Q ImpRes.

6. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 3, the gradient elution is performed using a linear gradient of 0-0.3 M NaCl solution, with a concentration change rate of 0.02-0.04 M / CV.

7. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 4, the filler for bionic ligand analysis is a bionic ligand filler, which is prepared from serum type ligand and sedimentation gel.

8. The process for purifying a pneumococcal monovalent polysaccharide according to claim 7, characterized in that: The serotype ligands are specifically: serotype 1, 2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 20, 22F, and 33F ligands are shown in SEQ ID NO.1, the serotype 6B ligand is shown in SEQ ID NO.2; the serotype 19F ligand is shown in SEQ ID NO.3; and the serotype 23F ligand is shown in SEQ ID NO.

4.

9. The process for purifying a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In step 4, the flow-through mode collection includes equilibration, sample loading, and elution collection.

10. The purification process of a pneumococcal monovalent polysaccharide according to claim 1, characterized in that: In the step 4, the collection standard is UV260<50mAU.

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