CN112646050B

CN112646050B - Purification process of pneumococcal polysaccharide

Info

Publication number: CN112646050B
Application number: CN202110026708.4A
Authority: CN
Inventors: 马可; 朱卫华; 胡国伟; 李爽; 林彦斌; 王瑞峰; 杜琳
Original assignee: Anhui Zhifei Longcom Biopharmaceutical Co ltd; Chongqing Zhifei Biological Products Co Ltd; Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Current assignee: Anhui Zhifei Longcom Biopharmaceutical Co ltd; Chongqing Zhifei Biological Products Co Ltd; Beijing Zhifei Lvzhu Biopharmaceutical Co Ltd
Priority date: 2021-01-09
Filing date: 2021-01-09
Publication date: 2023-01-10
Anticipated expiration: 2041-01-09
Also published as: CN112646050A

Abstract

The invention discloses a process for purifying pneumococcal polysaccharide by ion exchange chromatography. The method comprises the following steps: the pneumococcus is subjected to fermentation culture, inactivation, clarification, ultrafiltration concentration and other steps to obtain a pneumococcus polysaccharide crude extract solution, and impurities in the purification process of pneumococcus capsular polysaccharide of different serotypes are removed through an ion exchange chromatography process to obtain the desired pneumococcus polysaccharide.

Description

Purification process of pneumococcal polysaccharide

Technical Field

The invention belongs to the technical field of biology, and relates to a purification process of pneumococcal polysaccharide.

Background

The chromatographic technique is one of the most effective means for separating and purifying biological macromolecules, has various separation mechanisms such as ion exchange, hydrophobicity, reversed phase, metal chelation, affinity, gel filtration and the like, can realize very high separation degree, has simple equipment, is convenient for automatic control, and can not generate the effects of heating, phase change and the like in the separation process, so the chromatographic technique is more and more widely applied to the actual separation and purification of the biological macromolecules. Ion exchange chromatography is a chromatography technology which takes an ion exchanger as a stationary phase and a specific solution containing ions as a mobile phase and separates various substances in a mixture by utilizing the difference of the combination of the ion exchanger on biomacromolecules needing to be separated.

Pneumococcal capsular polysaccharide is an effective antigen for preparing pneumococcal related vaccines. After the pneumococcal capsular polysaccharide is cultured by bacterial fermentation, the conventional purification process of the pneumococcal polysaccharide is obtained by phenol extraction and ethanol fractional precipitation purification. However, phenol has a strong corrosive action on skin and mucous membranes, can inhibit central nerves or damage liver and kidney functions, and can cause pollution to environmental water and atmosphere. Ethanol is a flammable and explosive hazardous chemical, has extremely high requirements on the design of a production plant and has great influence on the production cost. And the use of toxic, harmful and severely polluting organizing agents poses hazards to workers and the environment, so the development of innovative, safe and effective purification processes is urgently needed.

The purification process of the invention refers to that the pneumococcus is fermented and cultured, inactivated, clarified, ultrafiltered and concentrated to obtain crude and pure pneumococcus polysaccharide, and then impurities such as protein, nucleic acid and the like are removed through ion exchange chromatography to finally obtain the pneumococcus capsular polysaccharide.

Disclosure of Invention

The invention aims to provide a purification process of pneumococcal polysaccharide.

The invention mainly aims at the chromatography process, the medium used in chromatography is an anion exchange medium, and different diluents are used to achieve the purpose of removing impurities due to the properties of pneumococcal capsular polysaccharides of different serotypes; different sample loading amounts are used according to the properties of pneumococcal capsular polysaccharides of different serotypes, so that the chromatography efficiency is improved; and the cleaning procedure of the chromatography medium is optimized, and the service life of the chromatography medium is prolonged.

The pneumococcal polysaccharide prepared by the existing method has lower purity and is crude and pure polysaccharide. The invention further performs chromatography purification on the crude and pure polysaccharide to obtain the pneumococcal capsular polysaccharide with higher purity.

Specifically, the purification process of pneumococcal polysaccharide provided by the invention comprises the following steps:

(1) Preparing chromatographic equilibrium solution of 0.01mol/L to 0.2mol/L phosphate buffer solution, and the pH value range is 6.0 to 7.6.

(2) Ultrafiltering and concentrating crude pure polysaccharide solution of pneumococcus, ultrafiltering with membrane with molecular weight cutoff of 10-100 Kd, dialyzing with chromatographic equilibrium liquid, and collecting ultrafiltered concentrate after dialysis.

(3) And (4) balancing the chromatographic column by using a chromatographic balance liquid.

(4) And (4) loading, eluting by using an equilibrium buffer solution, monitoring an ultraviolet absorption peak at 206nm, and collecting a flow-through peak.

Wherein the ion exchange medium used for chromatography is ion exchange medium DEAE Sepharose FF.

Wherein, the pneumococcal crude pure polysaccharide can be purchased in the market and can also be prepared according to the conventional method.

Here, a method for producing a crude pure polysaccharide from pneumococcus is exemplified, but it is needless to say that it can be produced by other methods.

Wherein, the preparation steps of the pneumococcal crude pure polysaccharide are as follows:

any one of the 24 serotypes of streptococcus pneumoniae is fermented, the pneumococcus is inactivated, fermentation liquor is clarified, the fermentation liquor is subjected to ultrafiltration concentration, CTAB is added to form precipitates with polysaccharide, the precipitates are collected centrifugally, depolymerization is performed through sodium chloride, supernatant is collected centrifugally, ultrafiltration concentration and buffer replacement are performed, the polysaccharide is purified by chromatography, ultrafiltration desalting is performed on water for injection, and freeze-drying is performed to obtain the crude and pure streptococcus pneumoniae polysaccharide finally.

The purification process is suitable for the streptococcus pneumoniae

serotypes including types

1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 12F, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

The streptococcus pneumoniae serum is divided into 24 serotypes, and different purification processes are adopted according to different types of serum.

The invention screens 24 serotypes, and the serotypes are divided into 6 groups according to the concentration of buffer and the loading volume, and the corresponding 6 different purification methods, and the specific screening method is detailed in the embodiment.

Group 1, a process for purifying crude pure polysaccharides of streptococcus pneumoniae

serotypes

2, 9N, 9V, 10A, 12F, 14, 15B, 17F, 18C, 20, 22F and 23F, comprising the steps of:

(1) Preparing a chromatographic equilibrium solution: the concentration is 0.1mol/L phosphate buffer solution, and the conductivity value of the equilibrium solution is detected and ranges from 20 +/-2 mS/cm; detecting the pH value of the equilibrium solution within the range of 7.50 +/-0.10;

(2) Ultrafiltering and concentrating the crude pure polysaccharide solution, wherein the molecular weight cut-off of an ultrafiltration membrane is 100Kd, dialyzing with chromatographic equilibrium liquid until the electric conductance is consistent, and collecting the ultrafiltration concentrate after dialysis;

(3) Balancing the chromatographic column with chromatographic equilibrium liquid until the conductivity is stable;

(4) The sample loading amount of the chromatography is not more than 0.25 column volume (hereinafter abbreviated as CV), the flow-through peak is eluted by using an equilibrium buffer, the ultraviolet absorption peak at 206nm is monitored, and the flow-through peak, namely the pneumonia polysaccharide chromatography component, is collected.

Group 2, a process for purifying crude pure polysaccharides of streptococcus pneumoniae serotypes 4, 8, 19A and 19F, comprising the steps of:

(1) The chromatographic equilibrium solution is 0.2mol/L phosphate buffer solution, and the conductivity value of the dialysate is detected within the range of 40 +/-4 mS/cm; detecting the pH value of the dialysate, wherein the range is 7.50 +/-0.10;

(3) Balancing the chromatographic column by using a chromatographic balance liquid until the conductivity is stable;

(4) The sample loading amount of the chromatographic equilibrium liquid is not more than 0.25CV, the flow-through peak is eluted by the chromatographic equilibrium liquid, the ultraviolet absorption peak of 206nm is monitored, and the flow-through peak, namely the pneumonia polysaccharide chromatographic component, is collected.

Group 3, a process for purifying crude pure polysaccharides of streptococcus pneumoniae serotypes 6A and 6B, comprising the steps of:

(1) Preparing a chromatographic equilibrium solution which is 0.1mol/L phosphate buffer solution, detecting the conductivity value of the equilibrium solution within the range of 20 +/-2 mS/cm, and detecting the pH value of the equilibrium solution within the range of 7.50 +/-0.10; preparing a 0.2mol/L phosphate buffer solution of chromatographic eluent, detecting the conductivity value of the eluent within the range of 40 +/-4 mS/cm, and detecting the pH value of the eluent within the range of 7.50 +/-0.10;

(3) And (5) balancing the chromatographic column by using a chromatographic equilibrium liquid until the conductivity is stable.

(4) And (3) keeping the sample loading amount to be not more than 0.25CV, keeping the flow rate, eluting by using chromatographic equilibrium liquid for 1CV, then eluting by using chromatographic eluent for 2CV, monitoring an ultraviolet absorption peak at 206nm, and collecting an elution peak, namely the pneumonia polysaccharide chromatographic component.

Group 4, a process for purifying crude pure polysaccharides of streptococcus pneumoniae serotypes 1, 5, 7F and 33F, comprising the steps of:

(2) Ultrafiltering and concentrating the crude pure polysaccharide solution, wherein the molecular weight cut-off of an ultrafiltration membrane is 100Kd, dialyzing by using chromatographic equilibrium liquid until the electric conductance is consistent, and collecting the ultrafiltration concentrated solution after the dialysis is finished;

(4) And (3) eluting the flow-through peak by using an equilibrium buffer solution with the sample loading amount not exceeding 0.5CV, monitoring the ultraviolet absorption peak at 206nm, and collecting the flow-through peak, namely the pneumonia polysaccharide chromatographic component.

Group 5, the purification process for streptococcus pneumoniae serotype 3 crude pure polysaccharide, comprising the following steps:

(4) The sample loading amount of the chromatographic equilibrium liquid is not more than 4CV, the chromatographic equilibrium liquid is used for eluting the flow-through peak, the ultraviolet absorption peak of 206nm is monitored, and the flow-through peak, namely the pneumonia polysaccharide chromatographic component, is collected.

Group 6, a process for purifying a crude and pure polysaccharide of streptococcus pneumoniae serotype 11A, comprising the steps of:

(4) The sample loading amount of the chromatographic equilibrium liquid is not more than 0.5CV, the chromatographic equilibrium liquid is used for eluting the flow-through peak, the ultraviolet absorption peak at 206nm is monitored, and the flow-through peak, namely the pneumonia polysaccharide chromatographic component, is collected.

The prepared polysaccharide is detected, and the detection method of the protein content and the nucleic acid content is referred to Chinese pharmacopoeia.

Compared with the existing purification process, the purification process is safer and more effective, and the efficiency of chromatography is optimized by using different sample loading amounts according to different serotypes.

Drawings

FIG. 1 is a chromatogram of pneumococcal polysaccharide type 7F from example 1.

FIG. 2 is a chromatogram of pneumococcal polysaccharide type 6A of example 2.

FIG. 3 is a chromatogram of pneumococcal polysaccharide type 8 from example 3.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.

The main instruments and equipment involved in the following examples:

model FE30 conductivity meter available from METTLER TOLEDO; ultrafiltration membranes were purchased from MILLIPORE corporation; the chromatography system was AKTA PURE from GE.

Example 1: purification method of streptococcus pneumoniae serotype 7F crude and pure polysaccharide

The purification is carried out by 500ml of streptococcus pneumoniae serotype 7F crude pure polysaccharide, and comprises the following steps:

(1) 0.1mol/L phosphate buffer solution is prepared as an equilibrium solution, the conductivity value is detected to be 20.46mS/cm, and the pH value is detected to be 7.53.

(2) And (3) performing ultrafiltration concentration on 500ml of a sample before chromatography of the streptococcus pneumoniae serotype 7F to 100ml, maintaining the volume, performing dialysis by using chromatographic equilibrium liquid, and collecting 100ml of ultrafiltration concentrate after the dialysis is finished.

(3) The column was equilibrated with equilibration solution and monitored to stabilize the conductivity at 20.52mS/cm.

(4) And (3) carrying out chromatography sample loading of 5ml, keeping the flow rate at 5ml/min, eluting the flow-through peak by using 0.1mol/L phosphate buffer solution, monitoring the ultraviolet absorption peak at 206nm, and collecting the flow-through peak.

(5) Samples are collected, freeze-dried and detected after ultrafiltration and desalination by water for injection, and the detection results are shown in table 1.

Table 17F pneumococcal polysaccharide recovery and physicochemical indices:

example 2: purification method of streptococcus pneumoniae serotype 6A crude and pure polysaccharide

The purification is carried out by 500ml of streptococcus pneumoniae serotype 6A crude pure polysaccharide, and comprises the following steps:

(1) 0.1mol/L phosphate buffer solution is prepared as equilibrium liquid, the electric conductivity value is detected to be 20.46mS/cm, and the pH value is 7.53.

(2) 0.2mol/L phosphate buffer solution is prepared as eluent, the conductance value is detected to be 40.33mS/cm, and the pH value is 7.50.

(3) The ultrafiltration membrane has a molecular weight cutoff of 100Kd, 500ml of feed liquid of streptococcus pneumoniae serotype 6A crude sugar is concentrated to 100ml, the volume is maintained, equilibrium liquid is used for dialysis, and 100ml of ultrafiltration concentrate is collected after the dialysis is finished.

(4) The column was equilibrated with equilibration solution and monitored to stabilize the conductivity at 20.39mS/cm.

(5) 5ml of chromatography sample feeding concentrated solution, the flow rate is 5ml/min, the flow rate is kept, 25ml of chromatography sample feeding concentrated solution is eluted by 0.1mol/L phosphate buffer solution, then 0.2mol/L phosphate buffer solution is used for elution, the ultraviolet absorption peak of 206nm is monitored, and the elution peak is collected.

(6) Samples were collected, desalted by ultrafiltration with water for injection, and then freeze-dried for detection, and the detection results are shown in table 2. Table 2 6a pneumococcal polysaccharide recovery and physicochemical indices:

example 3: purification method of streptococcus pneumoniae serotype 8 crude and pure polysaccharide

The purification is carried out by 500ml of streptococcus pneumoniae serotype 8 crude pure polysaccharide, and comprises the following steps:

(1) 0.2mol/L phosphate buffer solution is prepared as a balance liquid, the detection conductance value is 40.46mS/cm, and the pH value is 7.50.

(2) 500ml of crude streptococcus pneumoniae serotype 7F saccharide is ultrafiltered and concentrated to 100ml, the volume is maintained, chromatographic equilibrium liquid is used for dialysis, and 100ml of ultrafiltered concentrated liquid is collected after the end of dialysis.

(3) The column was equilibrated with 0.2mol/L phosphate buffer solution and monitored to a conductivity of 40.52mS/cm.

(4) Chromatography is carried out on the sample with the flow rate of 5ml/min, the flow rate is maintained, 0.2mol/L phosphate buffer solution is used for elution, the ultraviolet absorption peak at 206nm is monitored, and the flow-through peak is collected.

(5) Samples were collected, desalted by ultrafiltration with water for injection, and then freeze-dried for detection, and the detection results are shown in table 3.

Table 3 pneumococcal polysaccharide recovery rate and physicochemical index conditions:

test example 1 screening of equilibration buffer and elution buffer

The invention uses buffer solutions with different concentrations as a balance buffer solution and an elution buffer solution for purifying polysaccharide of 24 serotypes, and screens the polysaccharide chromatography buffer solution concentrations of different serotypes: equilibrating and loading Streptococcus pneumoniae serotypes 1, 2, 5, 7F, 9N, 9V, 10A, 12F, 14, 15B, 17F, 18C, 20, 22F, 23F and 33F with 0.1mol/L phosphate buffer and eluting with 0.1mol/L phosphate buffer; equilibrating and loading type 3, type 4, type 11A, type 19A and type 19F with 0.2mol/L phosphate buffer, eluting with 0.2mol/L phosphate buffer; the type 6A and type 6B equilibration and loading were eluted with 0.1mol/L phosphate buffer and 0.2mol/L phosphate buffer.

The specific screening process, taking streptococcus pneumoniae serotype 33F as an example, 500ml of 33F crude pure polysaccharide is purified, and comprises the following steps:

(1) 10mmol/L, 50mmol/L, 75mmol/L, 0.1mol/L and 0.2mol/L phosphate buffer solutions are respectively prepared as chromatographic equilibrium solutions, and the detection conductances are respectively 2.09mS/cm, 10.93mS/cm, 15.02mS/cm, 20.46mS/cm and 40.33mS/cm.

(2) 500ml of feed liquid of streptococcus pneumoniae serotype 33F crude sugar is ultrafiltered and concentrated to 100ml, the volume is maintained, 10mmol/L phosphate buffer solution is used for dialysis, and 100ml of ultrafiltration concentrate is collected after the end of dialysis.

(3) Adjusting the electric conductivity of the ultrafiltration concentrated solution to be consistent with that of different chromatographic equilibrium solutions respectively.

(4) And (4) carrying out sample chromatography respectively, and eluting each sample by using a corresponding chromatographic equilibrium solution.

(5) The UV absorption peak at 206nm was monitored and the flow-through peak was collected for all 4 samples except 10mmol/L phosphate buffer solution.

(6) After the flow-through peaks of each different chromatographic sample are collected, the samples are respectively subjected to ultrafiltration desalting by water for injection and then freeze-drying detection, and the detection results are shown in table 4.

The results show that the chromatography recovery rate is highest and the protein content and the nucleic acid content are lowest when the phosphate buffer solution of 0.1mol/L is used as the chromatographic equilibrium liquid. For form 33F, preferably, 0.1mol/L phosphate buffer is used as chromatographic equilibration and elution. Other serotypes were optimally screened for elution buffer using similar methods.

TABLE 4, 33F pneumococcal polysaccharide recovery rate and physicochemical index conditions

Test example 2 sample Loading amount screening

The invention screens the polysaccharide loading amount of 24 serotypes to achieve the optimal chromatography efficiency: serotype 2, type 4, type 6A, type 6B, type 8, type 9N, type 9V, type 12F, type 10A, type 12F, type 14, type 15B, type 17F, type 18C, type 19A, type 19F, type 20, type 22F and type 23F streptococcus pneumoniae chromatographic loading volumes are no greater than 0.25CV; sample volumes of form 1, 5, 7F, 11A and 33F are no greater than 0.5CV; the sample volume of type 3 is not more than 4CV.

The specific screening process, taking streptococcus pneumoniae serotype 11A as an example, 500ml of crude pure polysaccharide is purified, and the method comprises the following steps:

(1) Preparing 0.2mol/L phosphate buffer solution, detecting that the conductance value is 40.46mS/cm, and the pH value is 7.50.

(2) 500ml of crude streptococcus pneumoniae serotype 11A sugar is taken, ultrafiltration concentration is carried out to 100ml, the volume is maintained, chromatography equilibrium liquid is used for dialysis, and 100ml of ultrafiltration concentrated liquid is collected after the end of dialysis.

(3) The column was equilibrated with 0.2mol/L phosphate buffer solution and conductivity was monitored for stability.

(4) The ratios of the different sample loading amounts to the Column Volume (CV) were respectively designated as 0.25CV, 0.5CV, 0.75CV and 1CV.

(5) The same flow rate was maintained, elution was performed using 0.2mol/L phosphate buffer, the UV absorption peak at 206nm was monitored, and the flow-through peak was collected.

(6) The flow-through peak was subjected to lyophilization detection after ultrafiltration and desalting with water for injection, and the detection results are shown in table 5.

As a result, the protein content was found to be unacceptable at the loading of 1CV and acceptable at the loading of 0.75CV, but the protein content was found to be significantly higher than the protein content at the loading of 0.25CV and the protein content at the loading of 0.5CV, and it was found that the maximum loading of 11A was not more than 0.5CV. Other serotypes were optimally screened for loading volume using similar methods.

TABLE 5 11A pneumococcal polysaccharide recovery rate and physicochemical index conditions

Claims

1. A method for purifying pneumococcal polysaccharide, wherein the pneumococcal polysaccharide is serotype 33F streptococcus pneumoniae, and the method comprises the following steps:

(1) Preparing a chromatographic equilibrium solution: the concentration is 0.1mol/L phosphate buffer solution, and the conductivity value of the equilibrium liquid is detected and ranges from 20 +/-2 mS/cm; detecting the pH value of the equilibrium solution within the range of 7.50 +/-0.10;

(3) Balancing the chromatographic column with chromatographic equilibrium liquid until the conductivity is stable, wherein the ion exchange medium used for chromatography is an ion exchange medium DEAE Sepharose FF;