CN113603804B - Refining method of streptococcus pneumoniae capsular polysaccharide - Google Patents
Refining method of streptococcus pneumoniae capsular polysaccharide Download PDFInfo
- Publication number
- CN113603804B CN113603804B CN202111019545.3A CN202111019545A CN113603804B CN 113603804 B CN113603804 B CN 113603804B CN 202111019545 A CN202111019545 A CN 202111019545A CN 113603804 B CN113603804 B CN 113603804B
- Authority
- CN
- China
- Prior art keywords
- streptococcus pneumoniae
- capsular polysaccharide
- sample solution
- pneumoniae capsular
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
- A61K39/092—Streptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The application belongs to the technical field of biology, and particularly discloses a method for refining streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: preparing a sample solution containing streptococcus pneumoniae capsular polysaccharide, flocculating, heating and acid precipitating, centrifuging, filtering, ultrafiltering and the like. The application has at least one of the following beneficial effects: according to the refining method of the streptococcus pneumoniae capsular polysaccharide, the refined streptococcus pneumoniae capsular polysaccharide solution far higher than the standard of European pharmacopoeia can be prepared by conventional methods such as flocculation, temperature rise acid precipitation, centrifugation, filtration and ultrafiltration, and purification steps such as chromatographic chromatography and carbon filtration are not needed in the refining method, so that the recovery rate is improved, the process steps are shortened, and the large-scale industrial production is facilitated.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for refining streptococcus pneumoniae capsular polysaccharide.
Background
Pneumococcal disease has been one of the major public health problems worldwide. Depending on the site of infection with Streptococcus pneumoniae, pneumococcal disease can be classified into Invasive Pneumococcal Disease (IPD) and non-invasive pneumococcal disease (NIPD). IPD refers to infection caused by streptococcus pneumoniae invading originally sterile sites and tissues, mainly including meningitis, septicemia, bacterial pneumonia and the like, and has a high incidence in children under 5 years of age, especially infants under 2 years of age. Maternal antibodies gradually disappear from 6 months of age in infants, and thus, the period from 6 months of age to 2 years of age is the period of highest incidence of IPD in children.
At present, more than 90 serotypes of streptococcus pneumoniae exist, but more than 80% of IPD of all age groups in the world are related to 30 serotypes, and years of research and clinical application prove that capsular polysaccharide of streptococcus pneumoniae can induce specific antibodies as a vaccine and has good immune protection effect on corresponding streptococcus pneumoniae. Capsular polysaccharides of different serotypes of streptococcus pneumoniae induce poor cross-protection of antibodies and it is therefore often necessary to use combinations of streptococcus pneumoniae polysaccharides of different serotypes as vaccines to prevent the pathogenicity of major streptococcus pneumoniae.
In order to meet the purity of capsular polysaccharide required by vaccine preparation, fermentation broth must be treated through multiple purification steps to remove impurities such as protein and nucleic acid in polysaccharide extract, thereby avoiding side reactions possibly caused by subsequent vaccination. However, the conventional polysaccharide refining process has many and complicated steps, and the polysaccharide solution refined by using a simple process method mostly does not meet the standard of the european pharmacopoeia, and the refined polysaccharide solution meeting the standard of the european pharmacopoeia is required to be obtained and further processed by a single or a plurality of chromatographic steps. On one hand, the yield is influenced due to the property of the filler, the volume of the treated solution is small, and the amplification production is limited; on the other hand, the cost of scale production is increased.
Disclosure of Invention
Based on the method, the refined streptococcus pneumoniae capsular polysaccharide which reaches the standards of European pharmacopoeia can be prepared by conventional purification methods such as flocculation, temperature-rising acid precipitation, centrifugal filtration, ultrafiltration and the like.
The application is realized by the following scheme:
the application provides a refining method of streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: s1: preparing a sample solution containing streptococcus pneumoniae capsular polysaccharide; s2: flocculation: adding aluminum sulfate into the sample solution to enable the final concentration (m/v) of the aluminum sulfate to be 0.5-3.0%, and reacting to obtain a first sample solution; s3: heating and acid precipitation: adjusting the pH value of the first sample solution to 3.5-4.0, and then heating the acidified solution to 45-55 ℃ for reaction to obtain a second sample solution; or, firstly, heating the first sample solution to 45-55 ℃, then adjusting the pH value to 3.5-4.0, and reacting to obtain a second sample solution; s4: centrifuging and filtering: centrifuging, clarifying and filtering the second sample solution to obtain a third sample solution; s5: and (3) ultrafiltration: and carrying out ultrafiltration on the third sample solution by using an ultrafiltration membrane to obtain a refined selected streptococcus pneumoniae capsular polysaccharide solution.
According to the method, the refined streptococcus pneumoniae capsular polysaccharide solution meeting the standards of European pharmacopoeia can be obtained by conventional purification methods such as flocculation, temperature rise and acid precipitation, centrifugation, filtration and ultrafiltration, and the process steps such as chromatographic chromatography and carbon filtration are not needed.
In a particular embodiment of the present application, the final concentration of aluminum sulfate (m/v) is 0.5%, 1%, 1.5%, 2%, 2.5%, 3.0%, or the like.
In one embodiment of the present application, the pH is 3.5, 3.75 or 4.0.
In one embodiment of the present application, the temperature of the acidified solution is 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃ or 55 ℃.
In a specific embodiment of the present application, the sequence of the step S2 and the step S3 may be exchanged.
In one embodiment of the present application, the reaction time of the flocculation is 20-40 min. For example, the reaction time is 20min, 23min, 25min, 28min, 30min, 32min, 35min, 37min or 40 min.
In one embodiment of the present application, the acid used for the acid precipitation is any one of phosphoric acid, acetic acid, hydrochloric acid, sulfuric acid, or nitric acid.
In one embodiment of the present application, the reaction time in the temperature-rising acid precipitation is 45 to 80 min. For example, the reaction time is 45min, 50min, 55min, 60min, 65min, 70min, 75min or 80min, etc.
In one embodiment of the present application, the sample solution is a streptococcus pneumoniae fermentation broth.
In a specific embodiment of the present application, the preparation method further comprises step S0: and (3) cracking the streptococcus pneumoniae fermentation culture solution by using sodium deoxycholate.
In one embodiment of the present application, the processing time of step S0 is 45-60 min. For example, the treatment time is 45min, 48min, 50min, 52min, 55min, 58min, or 60 min.
In one embodiment of the present application, the preparation method further includes step S6: and (3) sterilizing and filtering the capsular polysaccharide solution.
In one embodiment of the present application, the clarification filtration is microfiltration or depth filtration.
In one embodiment of the present application, the membrane pore size of the microfiltration is 0.22 to 1.0 μm.
In one embodiment of the present application, the depth filtration has a filtration precision of 4.0 to 18.0 μm.
In one embodiment of the present application, the ultrafiltration membrane has a molecular weight cut-off of 30-100 kD.
In a specific embodiment of the present application, the solution used in ultrafiltration is any one of water for injection, buffer solution or saline solution or any combination of several of them.
In order to save the operation time, the solution selected for ultrafiltration in the application is water for injection and buffer solution.
In one embodiment of the present application, the buffer is a citrate-phosphate buffer.
The water for injection in the present application means water in accordance with the specifications under the term of water for injection in the chinese pharmacopoeia.
In a specific embodiment of the application, the serotype of streptococcus pneumoniae is selected from one or more of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 13, 14, 15B, 15C, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 25, 31 and 33F.
Another aspect of the application provides the use of the solutions of capsular polysaccharides of various serotypes of streptococcus pneumoniae obtained as described above for the preparation of vaccines.
The preparation method provided by the application can be used for refining 30 common streptococcus pneumoniae capsular polysaccharide solutions of different serotypes related to Invasive Pneumococcal Diseases (IPD), the preparation process is simple, the purification time is short, the recovery rate is high, the obtained refined capsular polysaccharide meets the European pharmacopoeia standard, the side effects generated by using the refined capsular polysaccharide as a vaccine are few, and the preparation method is particularly suitable for inoculation of children under 5 years old, and especially provides a material for production of infant vaccines under 2 years old.
The refining method of the streptococcus pneumoniae capsular polysaccharide provided by the application has at least one of the following beneficial effects:
according to the method for refining the streptococcus pneumoniae capsular polysaccharide, the refined streptococcus pneumoniae capsular polysaccharide solution far higher than the standard of European pharmacopoeia can be prepared by conventional methods such as flocculation, temperature rise and acid precipitation, centrifugation, filtration and ultrafiltration, and purification steps such as chromatographic chromatography and carbon filtration are not needed in the preparation method, so that the recovery rate is improved, the process steps are shortened, and the large-scale industrial production is facilitated.
Drawings
Fig. 1 is a flow chart of capsular polysaccharide refinement as provided in the examples of the present application.
Fig. 2 is another flow diagram of capsular polysaccharide refinement as provided in the examples of the present application.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The technical solutions of the present invention will be described clearly and completely below with reference to embodiments of the present invention, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
The following examples are presented by way of example of a fermentation broth of a selected serotype of streptococcus pneumoniae capsular polysaccharide.
(1) Cracking a fermentation culture solution containing the selected serotype streptococcus pneumoniae capsular polysaccharide by using sodium deoxycholate to obtain a sample solution;
(2) adding aluminum sulfate into the sample solution obtained in the step (1) to enable the final concentration (m/v) of the aluminum sulfate to be 0.5-3.0%, and reacting to obtain a first sample solution;
(3) heating and acid precipitation: adjusting the pH value of the first sample solution to 3.5-4.0, and then heating the acidified solution to 45-55 ℃ for reaction to obtain a second sample solution; alternatively, the first and second liquid crystal display panels may be,
heating the first sample solution to 45-55 ℃, and then adjusting the pH value to 3.5-4.0 for reaction to obtain a second sample solution;
s4: centrifuging and filtering: centrifuging the second sample solution, clarifying and filtering to obtain a third sample solution;
s5: and (3) ultrafiltration: and carrying out ultrafiltration on the third sample solution by using an ultrafiltration membrane to obtain the refined selected streptococcus pneumoniae capsular polysaccharide solution.
In the following examples, when sodium deoxycholate is used to lyse cells, subsequent purification can be performed by only 45-60min treatment; the buffer solution is citrate-phosphate buffer solution, wherein the molar concentration of citrate ions is 25mmol/L, and the molar concentration of phosphate ions is 10 mmol/L.
Polysaccharide recovery (%) = saccharide content in the final streptococcus pneumoniae capsular polysaccharide solution/saccharide content in the supernatant collected after centrifugation.
Polysaccharide yield (mg/L) = saccharide content in streptococcus pneumoniae capsular polysaccharide solution/volume of fermentation broth finally obtained.
Example 1: refinement of Streptococcus pneumoniae capsular polysaccharides type 3, 12F and 19F
In this example, the purification of streptococcus pneumoniae capsular polysaccharide type 3 is described as an example.
50L of streptococcus pneumoniae fermentation liquor 3 is added with sodium deoxycholate to make the final concentration (m/v) 0.1%, and the mixture is lysed for 60 minutes. Aluminum sulfate was slowly added to the lysate to a final concentration (m/v) of 0.5%, and stirred for 30 minutes. Adding acetic acid to adjust pH to 4.0, heating to 45 deg.C, stirring for 80min, centrifuging at 16000rpm for 60min, collecting supernatant, filtering with 0.45 μm microporous filter, and collecting filtrate. Concentrating the filtrate by 5 times, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 30kD, washing with citrate-phosphate buffer solution with volume 6 times of the concentrated solution, washing with water for injection until the conductivity of the filtrate is less than 20 μ S/cm, and sterilizing and filtering the obtained solution with 0.2 μm filter to obtain refined type 3 Streptococcus pneumoniae capsular polysaccharide solution.
The refining method of 12F type and 19F type streptococcus pneumoniae capsular polysaccharide is the same as 3 type, the detection is carried out according to the method of European pharmacopoeia, and each detection result is shown in table 1.
TABLE 1 detection results of serotypes 3, 12F and 19F
Note: "-" indicates no requirement.
As shown in Table 1, the detection results of the capsular polysaccharide solutions of streptococcus pneumoniae types 3, 12F and 19F refined by the method are all higher than the standard of European pharmacopoeia, the recovery rate of the polysaccharide is between 84.6 and 84.9 percent, the yield of the polysaccharide is higher than the requirement of industrial production (more than 200 mu g/ml), and the method is suitable for large-scale industrial production.
Example 2: refining of streptococcus pneumoniae capsular polysaccharides 1, 4, 6A, 6B and 7F
In this example, the purification of streptococcus pneumoniae capsular polysaccharide type 1 is described as an example.
50L of streptococcus pneumoniae capsular polysaccharide fermentation liquor 1 is added with sodium deoxycholate to ensure that the final concentration (m/v) is 0.1 percent, and the mixture is cracked for 45 minutes. Aluminum sulfate was slowly added to the lysate to a final concentration (m/v) of 1%, and stirred for 20 minutes. Adding phosphoric acid to adjust the pH value to 3.75, heating to 50 ℃, stirring for reaction for 45 minutes, cooling to room temperature, centrifuging at 16000rpm for 60 minutes, collecting supernate, filtering by a 0.45-micron micropore and collecting filtrate. Concentrating the filtrate by 6 times, ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 100kD, washing with citrate-phosphate buffer solution with volume 7 times of the concentrated solution, washing with water for injection until the conductivity of the filtrate is less than 20 μ S/cm, and filtering the obtained solution with 0.2 μm filter to remove bacteria and obtain refined Streptococcus pneumoniae capsular polysaccharide type 1.
The refining method of the streptococcus pneumoniae capsular polysaccharide types 4, 6A, 6B and 7F is the same as that of type 1, the detection is carried out according to the method of European pharmacopoeia, and each detection result is shown in a table 2.
TABLE 2 detection results of serotypes 1, 4, 6A, 6B, and 7F
Note: "-" indicates no requirement.
As shown in Table 2, the detection results of the streptococcus pneumoniae capsular polysaccharide solutions of types 1, 4, 6A, 6B and 7F refined by the method are all higher than the standard of European pharmacopoeia, the recovery rate of the polysaccharide is between 85.7 and 90.1 percent, the yield of the polysaccharide is higher than the requirement of industrial production, and the method is suitable for large-scale industrial production.
Example 3: refining of streptococcus pneumoniae capsular polysaccharide 5, 9V, 10A and 22F
In this example, the purification of streptococcus pneumoniae capsular polysaccharide type 5 is described as an example.
50L of streptococcus pneumoniae capsular polysaccharide type 5 fermentation liquor is added with sodium deoxycholate to ensure that the final concentration (m/v) is 0.1 percent, and the mixture is cracked for 50 minutes. Aluminum sulfate was slowly added to the lysate to a final concentration (m/v) of 1.5%, and stirred for 40 minutes. Adding nitric acid to adjust pH to 3.75, heating to 55 deg.C, stirring for 70min, cooling to room temperature, centrifuging at 16000rpm for 60min, collecting supernatant, deep filtering at 4.0-9.0 μm, and collecting filtrate. Concentrating the filtrate by 8 times, ultrafiltering by ultrafiltration membrane with molecular weight cutoff of 100KD, washing with citrate-phosphate buffer solution with volume 9 times of the concentrated solution, washing with water for injection until the conductivity of the filtrate is less than 20 μ S/cm, and sterilizing and filtering the obtained solution with 0.2 μm filter to obtain refined type 8 Streptococcus pneumoniae capsular polysaccharide solution.
The refining method of 9V type, 10A type and 22F type streptococcus pneumoniae capsular polysaccharide is the same as 5 type, the detection is carried out according to the method of European pharmacopoeia, and the detection results are shown in table 3.
TABLE 3 detection results of serotypes 5, 9V, 10A, and 22F
Note: "-" indicates no requirement.
As shown in Table 3, the detection results of the streptococcus pneumoniae capsular polysaccharide solutions of types 5, 9V, 10A and 22F refined by the method are all higher than the standard of European pharmacopoeia, the recovery rate of the polysaccharide is between 87.3 and 93.2 percent, the yield of the polysaccharide is higher than the requirement of industrial production, and the method is suitable for large-scale industrial production.
Example 4: refining of streptococcus pneumoniae capsular polysaccharide 8, 11A, 14 and 15B
In this example, the purification of streptococcus pneumoniae capsular polysaccharide type 8 is described as an example.
50L of Streptococcus pneumoniae fermentation broth 8, adding sodium deoxycholate to make the final concentration (w/v) 0.1%, and lysing for 55 minutes. Slowly adding aluminum sulfate into the lysate until the final concentration (w/v) is 2%, stirring for 25min, adding sulfuric acid to adjust the pH to 3.5, heating to 50 ℃, stirring for reaction for 60min, cooling to room temperature, centrifuging at 16000rpm for 60min, collecting the supernatant, performing deep filtration at 7.0-18.0 μm, and collecting the filtrate. Concentrating the filtrate by 7 times, ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 100KD, washing with 8 times volume of citrate-phosphate buffer solution, washing with water for injection until the filtrate conductivity is less than 20 μ S/cm, and sterilizing and filtering the obtained solution with 0.2 μm filter to obtain refined Streptococcus pneumoniae capsular polysaccharide type 5 solution.
The refining method of the streptococcus pneumoniae capsular polysaccharide types 11A, 14 and 15B is the same as that of type 8, the detection is carried out according to the method of European pharmacopoeia, and each detection result is shown in a table 4.
TABLE 4 detection results of serotypes 8, 11A, 14 and 15B
Note: "-" indicates no requirement.
As shown in Table 4, the detection results of the streptococcus pneumoniae capsular polysaccharide solutions of types 8, 11A, 14 and 15B refined by the preparation method are all higher than the European pharmacopoeia standard, the polysaccharide recovery rate is between 86.2 and 90.2 percent, the polysaccharide yield is higher than the industrial production requirement, and the preparation method is suitable for large-scale industrial production.
Example 5: refining of streptococcus pneumoniae capsular polysaccharides 18C, 20, 23F and 33F
In this example, the purification of the capsular polysaccharide of Streptococcus pneumoniae type 18C is described as an example.
50L of 18C Streptococcus pneumoniae fermentation broth was lysed by adding sodium deoxycholate to a final concentration (w/v) of 0.1% for 58 minutes. Adding hydrochloric acid to adjust the pH of the lysate to 3.75, heating to 55 ℃, stirring for reaction for 60 minutes, cooling to room temperature, slowly adding aluminum sulfate until the final concentration (w/v) is 3%, stirring for 20 minutes, centrifuging at 16000rpm for 60 minutes, taking the supernatant, filtering through a 0.45-micrometer micropore, and collecting the filtrate. Concentrating the filtrate by 10 times, ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 50KD, washing with citrate-phosphate buffer solution with volume 7 times of the concentrated solution, washing with water for injection until the conductivity of the filtrate is less than 20 μ S/cm, and sterilizing and filtering the obtained solution with 0.2 μm filter to obtain refined 18C type Streptococcus pneumoniae capsular polysaccharide solution.
The refining methods of the streptococcus pneumoniae capsular polysaccharide 20 types, 23F types and 33F types are the same as the method for refining the streptococcus pneumoniae capsular polysaccharide 18C types, the detection is carried out according to the method of European pharmacopoeia, and the detection results are shown in table 5.
TABLE 5 detection results of serotypes 18C, 20, 23F, and 33F
Note: "-" indicates no requirement.
As shown in Table 5, the detection results of the capsular polysaccharide solutions of streptococcus pneumoniae types 18C, 20, 23F and 33F refined by the preparation method are all higher than the standard of European pharmacopoeia, the recovery rate of the polysaccharide is between 84.6 and 89.8 percent, the yield of the polysaccharide is higher than the requirement of industrial production, and the preparation method is suitable for large-scale industrial production.
Example 6: refinement of Streptococcus pneumoniae capsular polysaccharides type 2, 9N, 17F and 19A
In this example, the purification of streptococcus pneumoniae capsular polysaccharide type 2 is described as an example.
50L of Streptococcus pneumoniae 2 fermentation broth was lysed for 52 minutes by adding sodium deoxycholate to a final concentration (w/v) of 0.1%. Slowly adding aluminum sulfate into the lysate until the final concentration (w/v) is 1.5%, stirring for 25 minutes, heating to 45 ℃, adding hydrochloric acid to adjust the pH to 3.5, stirring for reacting for 50 minutes, cooling to room temperature, centrifuging at 16000rpm for 60 minutes, collecting the supernatant, filtering through a 0.45-micrometer micropore, and collecting the filtrate. Concentrating the filtrate by 9 times, ultrafiltering with ultrafiltration membrane with cut-off molecular weight of 30KD, washing with citrate-phosphate buffer solution with volume 7 times of the concentrated solution, washing with water for injection until the conductivity of the filtrate is less than 20 μ S/cm, sterilizing the obtained solution with 0.2 μm filter, and filtering to obtain refined Streptococcus pneumoniae capsular polysaccharide type 2 solution.
The refining method of the 9N type, 17F type and 19A type streptococcus pneumoniae capsular polysaccharide solution is the same as that of the 2 type streptococcus pneumoniae capsular polysaccharide solution, the detection is carried out according to the method of European pharmacopoeia, and the detection results are shown in table 6.
TABLE 6 detection results of serotypes 2, 9N, 17F, and 19A
Note: "-" indicates no requirement.
As shown in Table 6, the detection results of the streptococcus pneumoniae capsular polysaccharide solutions type 2, type 9N, type 17F and type 19A refined by the preparation method are all higher than the European pharmacopoeia standard, the polysaccharide recovery rate is between 86.2% and 91.8%, the polysaccharide yield is higher than the industrial production requirement, and the preparation method is suitable for large-scale industrial production.
Other serotypes of streptococcus pneumoniae capsular polysaccharides are prepared as in types 13, 15C, 16, 24F, 25 and 31 by the same method as in example 1.
Example 7: comparison of different flocculants
The inventors of the present application explored the effect of different flocculants on the refining of various types of streptococcus pneumoniae capsular polysaccharides. The examples of this example are type 3 in example 1, type 4 in example 2, type 5 in example 3, type 8 in example 4, type 18C in example 5 and type 19A streptococcus pneumoniae capsular polysaccharide in example 6.
The refining methods of the streptococcus pneumoniae capsular polysaccharides of types 3, 4, 5, 8, 18C and 19A are the same as the above, except that aluminum sulfate serving as a flocculating agent is replaced by potassium aluminum sulfate, the detection is carried out according to the method of European pharmacopoeia, and the experimental results are shown in Table 7.
TABLE 7 comparison of different flocculants
As can be seen from Table 7, in the preparation of various Streptococcus pneumoniae capsular polysaccharides, the protein content and/or the nucleic acid content of the polysaccharide solution refined by using potassium aluminum sulfate as a flocculant do not meet the European pharmacopoeia standards, while the protein content and/or the nucleic acid content of the polysaccharide solution refined by using aluminum sulfate as a flocculant meet the European pharmacopoeia standards.
Example 8: comparison of different cleavage Agents
The inventors of the present application explored the effect of different lytic agents on the purification of various types of streptococcus pneumoniae capsular polysaccharides. In this example, the capsular polysaccharide from Streptococcus pneumoniae type 14 in example 4, 23F in example 5, and 9N in example 6 are exemplified.
9N type, 14 type, 23F streptococcus pneumoniae capsular polysaccharide refining method is the same as above, except that sodium deoxycholate as a cracking agent is replaced by N-lauroyl sarcosine sodium (NLS), and detection is carried out according to the method of European pharmacopoeia, and the experimental results are shown in Table 8.
TABLE 8 comparison of different flocculants
As can be seen from Table 8, in the preparation of various Streptococcus pneumoniae capsular polysaccharides, the protein content and the nucleic acid content of the polysaccharide solution refined by using sodium deoxycholate as a cracking agent are relatively lower, and the polysaccharide yield is relatively higher.
Example 9: comparison of different acid precipitation temperatures
The inventors of the present application explored the effect of different acid precipitation times on the purification of various types of streptococcus pneumoniae capsular polysaccharides. In this example, the streptococcus pneumoniae capsular polysaccharide type 9V is exemplified.
50L of 9V streptococcus pneumoniae capsular polysaccharide fermentation liquor is added with sodium deoxycholate to ensure that the final concentration (m/V) is 0.1 percent, and the mixture is cracked for 50 minutes. Slowly adding aluminum sulfate into the lysate until the final concentration (m/v) is 1.5%, stirring for 40 minutes, adding sulfuric acid to adjust the pH to 3.75, dividing the solution into 5 parts, heating to 37 ℃, 45 ℃, 50 ℃, 55 ℃ or 65 ℃, stirring for reaction for 70 minutes, cooling to room temperature, centrifuging for 8 minutes at 12000g, collecting the supernatant, filtering through a 0.45-micrometer micropore and collecting the filtrate. Ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 100KD, washing with citrate-phosphate buffer solution with 9 times volume of filtrate, washing with water for injection until the conductivity of filtrate is less than 20 μ S/cm, sterilizing and filtering the obtained solution with 0.2 μm filter to obtain refined 9V type Streptococcus pneumoniae capsular polysaccharide solution, and detecting according to European pharmacopoeia method, wherein the test results are shown in Table 9.
TABLE 9 comparison of different acid precipitation temperatures
As can be seen from table 9, when the acid precipitation temperature is lower than 45 ℃, the polysaccharide recovery rate is lower than 65%, which is not favorable for industrial mass production; when the acid precipitation temperature is higher than 55 ℃, although the content of protein and nucleic acid meets the standard, the molecular size does not meet the standard of European pharmacopoeia, when the acid precipitation temperature is between 45 ℃ and 55 ℃, the recovery rate of the prepared 9V streptococcus pneumoniae capsular polysaccharide is over 87.8 percent, the preparation method is suitable for industrial large-scale production, and the nucleic acid, the protein, the molecular size and the like (other indexes such as total nitrogen, phosphorus and the like are not given) all meet the standard of pharmacopoeia.
When the acid precipitation temperature is 45-55 ℃, the recovery rate of other types of streptococcus pneumoniae capsular polysaccharide is over 84.6 percent, the method is suitable for large-scale industrial production, and the nucleic acid, the protein, the molecular size and the like all accord with pharmacopoeia standards.
Example 10: comparison of different pH values of acid precipitation
The inventors of the present application explored the effect of different acid precipitation pH values on the purification of different types of streptococcus pneumoniae capsular polysaccharides. In this example, the capsular polysaccharide of Streptococcus pneumoniae type 9V is exemplified.
50L of 9V streptococcus pneumoniae capsular polysaccharide fermentation liquor is added with sodium deoxycholate to ensure that the final concentration (m/V) is 0.1 percent, and the mixture is cracked for 50 minutes. Aluminum sulfate was slowly added to the lysate to a final concentration (m/v) of 1.5%, and stirred for 40 minutes. Dividing the obtained sample into 5 parts in equal parts, adding sulfuric acid to adjust the pH value to 3.0, 3.5, 4.0, 4.5 and 5.0, respectively heating to 55 ℃, stirring for reaction for 70 minutes, cooling to room temperature, centrifuging for 8 minutes at 12000g, collecting supernatant, filtering by a 0.45-micron micropore and collecting filtrate. Ultrafiltering with ultrafiltration membrane with molecular weight cutoff of 100KD, washing with citrate-phosphate buffer solution with volume 9 times of filtrate, washing with water for injection until the conductivity of filtrate is less than 20 μ S/cm, sterilizing the obtained solution with 0.2 μm filter, filtering to obtain refined 9V type Streptococcus pneumoniae capsular polysaccharide solution, and detecting according to European pharmacopoeia method, wherein the test results are shown in Table 10.
TABLE 10 comparison of different acid-precipitation pH values
As can be seen from table 10, when the pH of the acid precipitation is less than 3.5, the polysaccharide recovery rate decreases; when the pH value of the acid precipitation is more than 4, the protein content and/or the nucleic acid content do not accord with the standard of European pharmacopoeia; when the pH value of acid precipitation is 3.5-4.0, the recovery rate of the prepared 9V streptococcus pneumoniae capsular polysaccharide is more than 85.2 percent, the method is suitable for industrial large-scale production, and the sizes of nucleic acid, protein, molecules and the like (other indexes such as total nitrogen, phosphorus and the like are not given) all accord with pharmacopoeia standards.
When the pH value of the acid precipitation is 3.5-4.0, the recovery rate of other types of streptococcus pneumoniae capsular polysaccharide is over 84.6 percent, the method is suitable for large-scale industrial production, and the sizes of nucleic acid, protein, molecules and the like all accord with pharmacopoeia standards.
In conclusion, in the refining method of the streptococcus pneumoniae capsular polysaccharide, aluminum sulfate is selected as a flocculating agent in the preparation process, the temperature range is controlled to be 45-55 ℃, the pH value is controlled to be 3.5-4.0, and various types of streptococcus pneumoniae capsular polysaccharide solutions meeting pharmacopoeia standards can be prepared, namely, when various streptococcus pneumoniae capsular polysaccharide solutions are industrially prepared, only a sample solution of the selected serotype streptococcus pneumoniae capsular polysaccharide needs to be replaced without adjusting preparation process parameters.
The specific embodiments are only for explaining the present application and are not limiting to the present application, and those skilled in the art can make modifications to the embodiments without inventive contribution as required after reading the present specification, but all the embodiments are protected by patent law within the scope of the claims of the present application.
Claims (6)
1. A refining method of streptococcus pneumoniae capsular polysaccharide is characterized by comprising the following steps:
s1: preparing a sample solution containing capsular polysaccharides of selected serotypes of streptococcus pneumoniae;
s2: flocculation: adding aluminum sulfate with the final concentration (m/v) of 0.5-3.0% into the sample solution for processing to obtain a first sample solution;
s3: heating and acid precipitation: adjusting the pH value of the first sample solution to 3.5-4.0, and then heating the acidified solution to 45-55 ℃ for treatment to obtain a second sample solution; alternatively, the first and second electrodes may be,
heating the first sample solution to 45-55 ℃, and then adjusting the pH value to 3.5-4.0 for treatment to obtain a second sample solution;
s4: centrifuging and filtering: centrifuging the second sample solution, clarifying and filtering to obtain a third sample solution;
s5: and (3) ultrafiltration: performing ultrafiltration on the third sample solution by using an ultrafiltration membrane to obtain refined selected streptococcus pneumoniae capsular polysaccharide;
the sample solution is a streptococcus pneumoniae fermentation culture solution; the refining method further includes step S0: cracking the streptococcus pneumoniae fermentation culture solution by using sodium deoxycholate;
the serotype of streptococcus pneumoniae is selected from one or more of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 13, 14, 15B, 15C, 16, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 25, 31 and 33F.
2. The refining method of claim 1, wherein the order of the steps S2 and S3 can be reversed.
3. The refining method of claim 1, wherein the aluminum sulfate treatment time is 20-40 min.
4. The refining method according to claim 1, wherein the treatment time of the temperature-rising acid precipitation is 45-80 min.
5. The refining method as claimed in claim 1, further comprising step S6: and (3) sterilizing and filtering the capsular polysaccharide solution.
6. The refining process of claim 1, wherein the clarification filtration is microfiltration or depth filtration.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111019545.3A CN113603804B (en) | 2021-09-01 | 2021-09-01 | Refining method of streptococcus pneumoniae capsular polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111019545.3A CN113603804B (en) | 2021-09-01 | 2021-09-01 | Refining method of streptococcus pneumoniae capsular polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113603804A CN113603804A (en) | 2021-11-05 |
| CN113603804B true CN113603804B (en) | 2022-07-26 |
Family
ID=78342530
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111019545.3A Active CN113603804B (en) | 2021-09-01 | 2021-09-01 | Refining method of streptococcus pneumoniae capsular polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113603804B (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216780A (en) * | 1997-11-05 | 1999-05-19 | 中国科学院微生物研究所 | Tech. and process for extracting pullulan from fermentation liquor |
| CN101081296A (en) * | 2006-05-29 | 2007-12-05 | 北京民海生物科技有限公司 | Method for preparing b type haemophilus influenzae capsular polysaccharide and united vaccines thereof |
| CN102078604A (en) * | 2010-12-31 | 2011-06-01 | 北京民海生物科技有限公司 | Meningococcus capsular polysaccharide polyvalent multivalent conjugate vaccine, preparation method and application thereof |
| CN102660601A (en) * | 2012-04-17 | 2012-09-12 | 江苏康泰生物医学技术有限公司 | Method for rapidly purifying bacteria capsular polysaccharide |
| CN103772086A (en) * | 2014-01-10 | 2014-05-07 | 国家海洋局第三海洋研究所 | Pretreatment process for preparing fractions of marine microorganism small molecule metabolites |
| CN104530250A (en) * | 2014-12-11 | 2015-04-22 | 北京科兴生物制品有限公司 | Method for purifying pneumococcal capsular polysaccharide |
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN107082819A (en) * | 2017-01-22 | 2017-08-22 | 华兰生物工程股份有限公司 | A kind of purification process of streptococcus pneumoniae capsular polysaccharide |
-
2021
- 2021-09-01 CN CN202111019545.3A patent/CN113603804B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1216780A (en) * | 1997-11-05 | 1999-05-19 | 中国科学院微生物研究所 | Tech. and process for extracting pullulan from fermentation liquor |
| CN101081296A (en) * | 2006-05-29 | 2007-12-05 | 北京民海生物科技有限公司 | Method for preparing b type haemophilus influenzae capsular polysaccharide and united vaccines thereof |
| CN102078604A (en) * | 2010-12-31 | 2011-06-01 | 北京民海生物科技有限公司 | Meningococcus capsular polysaccharide polyvalent multivalent conjugate vaccine, preparation method and application thereof |
| CN102660601A (en) * | 2012-04-17 | 2012-09-12 | 江苏康泰生物医学技术有限公司 | Method for rapidly purifying bacteria capsular polysaccharide |
| CN103772086A (en) * | 2014-01-10 | 2014-05-07 | 国家海洋局第三海洋研究所 | Pretreatment process for preparing fractions of marine microorganism small molecule metabolites |
| CN104530250A (en) * | 2014-12-11 | 2015-04-22 | 北京科兴生物制品有限公司 | Method for purifying pneumococcal capsular polysaccharide |
| CN104998255A (en) * | 2015-06-30 | 2015-10-28 | 北京祥瑞生物制品有限公司 | Novel ACYW135 meningococcal conjugate vaccine and preparation method thereof |
| CN107082819A (en) * | 2017-01-22 | 2017-08-22 | 华兰生物工程股份有限公司 | A kind of purification process of streptococcus pneumoniae capsular polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| "Quantification of residual cetyltrimethylammonium bromide (CTAB) and";Suresh Beri 等;《Biologicals》;20210431;第22-27页 * |
| "肺炎链球菌血清型7F荚膜多糖结合物中游离多糖含量的测定";韩悦颖 等;《中国新药杂志》;20141130;第23卷(第21期);第2501-2505+2541页 * |
| 6B型肺炎球菌荚膜多糖-破伤风类毒素结合物中游离多糖测定方法的建立;刘爱萍等;《中国新药杂志》;20171230(第24期);第61-66页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113603804A (en) | 2021-11-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6825950B2 (en) | Shortened purification process for the production of capsular Streptococcus pneumoniae polysaccharides | |
| KR20080046821A (en) | Method for purifying hyaluronic acid | |
| CN113215210B (en) | Method for preparing sialic acid by adopting polysialic acid fermentation liquor | |
| Morais et al. | Purification of capsular polysaccharides of Streptococcus pneumoniae: traditional and new methods | |
| CN102660601B (en) | The method of rapid purification of bacterial capsular polysaccharide | |
| CN107835821A (en) | For the method from microbial capsular separation of polysaccharides protein and other impurities | |
| CN105396129B (en) | Inactivated vaccine produced by poliomyelitis attenuated strain | |
| CN113603804B (en) | Refining method of streptococcus pneumoniae capsular polysaccharide | |
| CN105601735A (en) | Intravenously injected cytomegalovirus human immune globulin and preparation method thereof | |
| CN103721249B (en) | Meningitis vaccine and preparation method thereof | |
| CN107043431B (en) | Purification method of bacterial capsular polysaccharide | |
| CN102660603B (en) | Method for rapid purification of bacterial capsular polysaccharide | |
| CN102660602B (en) | Method for rapidly purifying bacteria capsular polysaccharide | |
| US10435433B2 (en) | Polysaccharide purification for vaccine production using lytic enzymes, tangential flow filtration, and multimode chromatography | |
| US20200023050A1 (en) | Polysaccharide Purification for Vaccine Production using Lytic Enzymes, Tangential Flow Filtration, and Multimode Chromatography | |
| CN104530250A (en) | Method for purifying pneumococcal capsular polysaccharide | |
| CN102731670B (en) | Hib capsular polysaccharide purifying process | |
| CN110892076A (en) | Process for quickly and efficiently purifying serogroup X neisseria meningitidis capsular polysaccharide | |
| CN107936128B (en) | Simple and effective bacterial vaccine purification method | |
| CN116970095A (en) | Preparation method of pneumococcal capsular polysaccharide | |
| CN114853917A (en) | Methods for preparing pneumococcal capsular polysaccharide and pneumococcal vaccine | |
| CN117756958A (en) | Method for preparing streptococcus pneumoniae capsular polysaccharide or polysaccharide degradation thereof | |
| CN116693706A (en) | Purification method of streptococcus pneumoniae capsular polysaccharide | |
| CN117756959A (en) | Preparation method of streptococcus pneumoniae capsular polysaccharide | |
| BR112020000229B1 (en) | PURIFICATION OF POLYSACCHARIDES FOR VACCINE PRODUCTION USING LYTIC ENZYMES, TANGENTIAL FLOW FILTRATION AND MULTIMODAL CHROMATOGRAPHY |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |