CN-120607643-B - Purification process of pneumococcal monovalent polysaccharide

Abstract

The invention discloses a purification process of pneumococcal monovalent polysaccharide, and particularly relates to the technical field of biological medicine. The process comprises pretreatment, anion chromatography, composite chromatography and biomimetic ligand chromatography. The purification process comprises the steps of removing 30% of protein and nucleic acid by an acid precipitation process, performing crude extraction by adopting anion chromatography, removing more than 80% of protein and nucleic acid, performing crude extraction by adopting anion chromatography, treating large-volume feed liquid, performing fine purification by utilizing composite chromatography, removing residual protein, nucleic acid and endotoxin, concentrating polysaccharide with high loading, performing deep impurity removal by utilizing bionic ligand chromatography, and removing trace nucleic acid in a targeted manner, thereby obtaining the single polysaccharide with the recovery rate of more than 90%, the protein content of less than 0.1%, the nucleic acid content of less than 0.1%, the endotoxin content of less than 0.1EU/mL and the removal rate of more than 99%.

Inventors

• AN YOUCAI
• JIA CHUNYU
• SHI CHANGJUN
• ZONG JUAN

Assignees

• 江苏中慧元通生物科技股份有限公司
• 易慧生物技术(上海)有限公司

Dates

Publication Date

20260512

Application Date

20250724

Claims (2)

1. 1. A process for purifying a pneumococcal monovalent polysaccharide, said process comprising: step one, pretreatment Centrifuging pneumococcal single polysaccharide to remove thalli, concentrating feed liquid by adopting an ultrafiltration concentration process, preliminarily removing impurities comprising partial proteins and partial nucleic acids by adopting an acid precipitation process, and preprocessing acid precipitation centrifugate by adopting an ultrafiltration system into a chromatography balance system PBS to obtain preprocessed crude extract; Step two, anion chromatography Passing the pretreated crude extract through an anion chromatographic column, collecting feed liquid in a flow-through mode to obtain DEAE flow-through liquid, and switching to a low-conductivity Tris buffer system for ultrafiltration liquid exchange to obtain DEAE treatment liquid; the anion chromatographic packing adopts high-resolution weak anion adsorption packing DEAE Bestrose HP; the program of the stream through mode is as follows: balancing with 0.1-0.3M PBS, pH=7.0-7.4, balancing anion chromatographic column, and flowing at 120-180cm/h, and 5CV volume; sampling, namely collecting pretreated crude extract with the flow rate of 120-180cm/h and the volume of 15CV, wherein the UV206 is more than or equal to 300 mAU; washing with 0.1-0.3M PBS, pH=7.0-7.4, and washing anion chromatographic column at flow rate of 120-180cm/h and volume of 3CV; Regeneration, namely, 1-3M NaCl solution, the flow rate is 100cm/h, the volume is 2CV, and the U.V 280 is zeroed; step three, composite chromatography Loading the DEAE treatment solution into a sample for composite chromatography, and collecting the Capto eluent through impurity washing and gradient elution; The specific operation is as follows: A filler Capto Q ImpRes; equilibration, equilibration of the composite column with 20mM Tris-HCl, pH=7.4, flow rate 60cm/h, volume 5CV; loading, namely, the flow rate of DEAE treatment fluid is 30cm/h, the volume is up to 60 mg polysaccharide/mL filler, and the UV280 baseline is adopted; Washing impurities with Tris-HCl 20 mM at flow rate of 60cm/h and volume of 2CV, with UV260<50mAU; gradient elution, namely 0 to 0.3M NaCl, wherein the linear slope is 0.03M/CV, the flow rate is 30cm/h, the volume is 10CV, and the section of 0.15 to 0.25M NaCl is collected to obtain Capto eluent; Regeneration, namely, 1M NaCl solution, the flow rate is 100cm/h, the volume is 2CV, the UV280 is zeroed, and the flow rate is 0.1M NaOH; diluting Capto Q eluent with ultrapure water for 3 times to make conductance less than or equal to 3 mS/cm to obtain diluent; step four, analyzing the bionic matching base layer Diluting Capto eluent with ultrapure water, reducing electric conductivity, performing bionic ligand chromatography, collecting eluent in a flow-through mode, and performing ultrafiltration concentration, sterilization, filtration and preservation; The flow-through mode collection comprises balancing, loading, eluting and collecting; Wherein, the equilibrium adopts a 5CV equilibrium buffer solution and a 20mM Tris-HCl equilibrium chromatographic column, and if the serotype 23F type is adopted, 0.1 mM ZnCl 2 solution is added into the equilibrium solution, and the sample loading speed is 10cm/h; The bionic ligand chromatographic packing is prepared from a serotype ligand and a sedimentation gel, wherein the sedimentation gel is activated by epoxy groups to obtain an activated gel, and the activated gel is prepared with the serotype ligand to obtain the bionic ligand packing, the serotype ligand is specifically a serotype 1,2, 3, 4, 5, 6A, 7F, 8, 9V, 9N, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 20, 22F and 33F ligand shown as H 2 N-Gly-Arg-Trp-Pro-Ser-Lys-Thr-Tyr-COOH, the serotype 6B ligand shown as H 2 N-Gly-Cit-Trp-Ser-Ser-Tyr-COOH, the serotype 19F ligand shown as H 2 N-Arg-Trp-His-Gly-Ser-Tyr-NH 2 , and the serotype 23F ligand shown as H 2 N-Arg-Trp-His-Ser-Tyr-COOH; The specific preparation method of the filler comprises the following steps: Washing, namely taking 100mL times of settled gel, washing 3 times by using ultrapure water, carrying out vacuum filtration until the water content is less than or equal to 5%, immersing in 20% ethanol, and preserving at 4 ℃ for later use; Epoxy group activation, namely adding 200 mL of 1, 4-butanediol diglycidyl ether into 100 mL settled gel, dissolving 0.2M NaOH in an activating agent, oscillating at a constant temperature of 25 ℃ and 150 rpm, reacting for 4 hours, and flushing with ultrapure water, 0.1M acetic acid, pH=4.0 and 5 CV of 20% ethanol after the reaction to obtain activated gel; Polypeptide solution, serotype ligand 2 mg/mL, dissolved in 0.1M carbonate buffer, pH 8.5; pumping the activated gel, adding a polypeptide solution with twice the sedimentation volume of the gel, introducing nitrogen to remove oxygen, sealing, performing oscillation reaction at 40 ℃, maintaining the pH value of 8.5+/-0.2, adding 1M ethanolamine, oscillating at the pH value of = 9.0,25 ℃ for 2 hours, and cleaning sequentially with 6M urea 5CV, 0.1M acetic acid 5CV and 20% ethanol 5CV to obtain the bionic ligand filler.
2. 2. The process for purifying a monovalent polysaccharide according to claim 1, wherein in the first step, the acid precipitation process comprises adding 45-55% acetic acid to the ultrafiltration concentrate, adjusting pH, stirring and stabilizing pH, adjusting temperature, and standing.

Description

Purification process of pneumococcal monovalent polysaccharide Technical Field The invention relates to the technical field of biological medicine, in particular to a purification process of pneumococcal monovalent polysaccharide. Background Streptococcus inflammatory is a gram positive bacterium, with more than 90 serotypes, most of which are covered by a layer of capsular polysaccharide. The capsular polysaccharide evades recognition by the immune system and prevents phagocytosis of immune cells, thus enabling bacteria to reproduce in vivo and cause disease. Years of researches and clinic prove that the capsular polysaccharide of the streptococcus pneumoniae can induce specific antibodies as a vaccine, and has good immunoprotection effect on the corresponding streptococcus pneumoniae. The capsular polysaccharides of streptococcus pneumoniae of different serotypes differ in composition and structure, and thus it is often desirable to use a combination of polysaccharides of streptococcus pneumoniae of different serotypes as a vaccine to prevent the pathogenicity of the principal streptococcus pneumoniae. Streptococcus pneumoniae used in the preparation of polysaccharide vaccines produces a large number of metabolites in addition to capsular polysaccharides during fermentation culture. In order to meet the purity of capsular polysaccharide necessary for vaccine preparation, the fermentation broth must be treated by multiple purification steps to remove impurities such as proteins, nucleic acids, endotoxins and the like from crude polysaccharide products, thereby avoiding side reactions that may be caused by subsequent vaccination. It is common practice to use ethanol precipitation to remove protein contaminants from polysaccharides efficiently, but it is difficult to meet the purity requirements of vaccines for injection. In addition, the use of ethanol also causes problems in that 1) the use of ethanol requires fire protection facilities, and the cost of designing such facilities is high, 2) the amount of ethanol required is large, almost 4-6L of ethanol is required per liter of processing material, and the waste liquid treatment is very difficult, and 3) the re-dissolution of the treated polysaccharide is difficult, and freeze-drying and other steps are required. Chinese patent CN112646050A adopts a process for purifying pneumococcal polysaccharide by ion exchange chromatography, and adopts a method for purifying pneumococcal polysaccharide by ion exchange chromatography, but the method has low loading, the increase of the loading can obviously increase the contents of protein and nucleic acid, the removal rate is low, and the low loading can ensure the removal rate, but the processing capacity is insufficient and the efficiency is too low. Chinese patent CN107043431A, a purification method of bacterial capsular polysaccharide, uses composite ion exchange chromatography and hydroxyl phosphate gray matter salting out to carry out chromatography to obtain a purified capsular polysaccharide solution, but the recovery rate of the obtained product is only 60 to 80 percent by using composite chromatography alone, the protein content is more than 0.5 percent, the removal rate is lower, and the patent still faces the problem of small processing sample size. Disclosure of Invention Therefore, the invention provides a purification process of pneumococcal monovalent polysaccharide, which aims to solve the problems in the prior art. In order to achieve the above object, the present invention provides the following technical solutions: According to the invention there is provided a process for purifying a pneumococcal monovalent polysaccharide, said process comprising: step one, pretreatment After pneumococci are activated, subjected to expansion culture and fermentation culture in a fermentation tank, inactivating and harvesting polysaccharide, centrifuging to remove thalli, concentrating feed liquid by adopting an ultrafiltration concentration process, preliminarily removing impurities comprising part of protein and part of nucleic acid by adopting an acid precipitation process, and preprocessing acid precipitation centrifugate into a chromatography balance system PBS by adopting an ultrafiltration system to obtain preprocessed crude extract; Step two, anion chromatography Passing the pretreated crude extract through an anion chromatographic column, collecting feed liquid in a flow-through mode to obtain DEAE flow-through liquid, and switching to a low-conductivity Tris buffer system for ultrafiltration liquid exchange to obtain DEAE treatment liquid; step three, composite chromatography The initial concentrated polysaccharide solution is subjected to sample loading and composite chromatography, and is subjected to impurity washing and gradient elution, so that Capto eluent is obtained; step four, analyzing the bionic matching base layer Diluting Capto eluent with ultrapure water, reducing electric conductivity, perf