CN112608392B - Preparation of pneumococcal capsular polysaccharide - Google Patents
Preparation of pneumococcal capsular polysaccharide Download PDFInfo
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- CN112608392B CN112608392B CN202011545068.XA CN202011545068A CN112608392B CN 112608392 B CN112608392 B CN 112608392B CN 202011545068 A CN202011545068 A CN 202011545068A CN 112608392 B CN112608392 B CN 112608392B
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- 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
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- 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
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Disclosed is a process for preparing pneumococcal capsular polysaccharides comprising: providing a pneumococcal fermentation broth; separating the pneumococcal fermentation liquor to obtain a first supernatant; filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate; adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant; diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate; dissolving the first precipitate to obtain a first redissolution; adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide.
Description
FIELD
The present disclosure relates generally to the preparation of vaccines, and more particularly to the preparation of pneumococcal polysaccharide vaccines.
Background
Pneumococcus is a gram-positive coccus with a membrane, and is a main pathogenic bacterium causing diseases such as human pneumonia, meningitis, otitis media, bacteremia and the like. The diseases caused by pneumococcus have high morbidity and mortality, and the main susceptible population is infants under 5 years old. As the resistance of pneumococcus has increased, the prevention of infection by vaccination has become increasingly important.
SUMMARY
In one aspect, the disclosure relates to a process for preparing a pneumococcal capsular polysaccharide comprising: providing a pneumococcal fermentation broth; separating the pneumococcal fermentation liquor to obtain a first supernatant; filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate; adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant; diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate; dissolving the first precipitate to obtain a first redissolution; adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide; wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
In another aspect, the present disclosure relates to pneumococcal capsular polysaccharides prepared by a process comprising the steps of: providing a pneumococcal fermentation broth; separating the pneumococcal fermentation liquor to obtain a first supernatant; filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate; adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant; diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate; dissolving the first precipitate to obtain a first redissolution; adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide; wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that a concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
In yet another aspect, the present disclosure relates to a pneumococcal vaccine comprising a pneumococcal capsular polysaccharide prepared by a process comprising the steps of: providing a pneumococcal fermentation broth; separating the pneumococcal fermentation liquor to obtain a first supernatant; filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate; adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant; diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate; dissolving the first precipitate to obtain a first redissolution; adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide; wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexacosyltrimethylammonium bromide (CTAB) is from 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
Detailed description of the invention
In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.
Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".
As used in this disclosure and the appended claims, a singular reference of an element without a numerical designation includes a plural reference unless the context clearly dictates otherwise.
Reference throughout the specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that a particular reference element, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
It should be understood that, as used in the specification of the present disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Definition of
In the present disclosure, the term "pneumococcus (Streptococcus pneumoniae)" belongs to the genus Streptococcus, and is a gram-positive coccus having a membrane.
In the present disclosure, the term "capsular polysaccharide" refers to pneumococcal capsular polysaccharide.
In the present disclosure, the term "cetyltrimethylammonium bromide (CTAB)" refers to a compound having the structure:
in certain embodiments, the term "water for injection" refers to water that meets the requirements under the term water for injection in pharmacopoeia of the people's republic of china, 2020.
In certain embodiments, the term "serotype" refers to a specific type of pneumococcus identified by a serological response.
Detailed Description
In one aspect, the present disclosure relates to a process for preparing a pneumococcal capsular polysaccharide comprising:
providing a pneumococcal fermentation broth;
separating the pneumococcal fermentation liquor to obtain a first supernatant;
filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate;
adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant;
diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate;
dissolving the first precipitate to obtain a first redissolution;
adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide;
wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of the chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
In certain embodiments, a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.0% (w/v).
In certain embodiments, the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.5% to 1.0% (w/v).
In certain embodiments, illustrative examples of first solutions containing chloride ions that can be used in the present disclosure include, but are not limited to, sodium chloride solutions, potassium chloride solutions, calcium chloride solutions, and mixtures thereof.
In certain embodiments, the pneumococcal fermentation broth is sterilized and then separated to obtain a first supernatant.
In certain embodiments, a pneumococcal fermentation broth is sterilized, pH adjusted, and then separated to provide a first supernatant.
In certain embodiments, illustrative examples of substances that can be used to adjust the pH of a pneumococcal fermentation broth include, but are not limited to, organic and inorganic acids.
In certain embodiments, illustrative examples of organic acids that can be used to adjust the pH of a pneumococcal fermentation broth include, but are not limited to, acetic acid, hydrochloric acid, and phosphoric acid.
In certain embodiments, a pneumococcal fermentation broth is sterilized, adjusted to a pH of 3.0 to 5.0, and then separated to obtain a first supernatant.
In certain embodiments, illustrative examples of methods of separating the pneumococcal fermentation broth include, but are not limited to, filtration and centrifugation.
In certain embodiments, the pneumococcal fermentation broth is sterilized and allowed to stand at 2 ℃ to 8 ℃, and then centrifuged to collect the supernatant, which is subjected to ultrafiltration to obtain an ultrafiltration concentrate, i.e., the first filtrate.
In certain embodiments, the volume of sodium chloride solution used for ultrafiltration is 5 to 10 times the volume of the supernatant.
In certain embodiments, ultrafiltration is performed using a membrane having a pore size of 100 kD.
In certain embodiments, illustrative examples of membranes that can be used for ultrafiltration of the supernatant of a pneumococcal fermentation broth of the present disclosure include, but are not limited to, ultrafiltration membranes.
In certain embodiments, a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate, allowed to stand at 2 ℃ to 8 ℃, and centrifuged to provide a second supernatant.
In certain embodiments, the second supernatant is diluted 1.5 to 4 times its volume with water for injection to a sodium chloride concentration of 0.2 to 0.5mol/L, followed by addition of a hexachloro-trimethyl-ammonium bromide (CTAB) and centrifugation to give a first precipitate.
In certain embodiments, hexacosyltrimethylammonium bromide (CTAB) is added to the diluted second supernatant, allowed to stand at 2 to 8 ℃, and centrifuged to give a first precipitate.
In certain embodiments, the second supernatant is diluted with water for injection to a volume of 1.5 to 4 times its volume to a sodium chloride concentration of 0.2 to 0.5mol/L, followed by addition of a hexacosanyltrimethylammonium bromide (CTAB), standing at 2 to 8 ℃, and centrifugation to give the first precipitate.
In certain embodiments, the first precipitate is dissolved in a sodium chloride solution having a concentration of 0.6 to 1.2mol/L and stirred at 2 to 8 ℃ to provide a first reconstituted solution.
In certain embodiments, the first precipitate is dissolved in a sodium chloride solution having a concentration of 1.0mol/L, preferably 1.0mol/L, and stirred at 2 ℃ to 8 ℃ to provide a first reconstituted solution.
In certain embodiments, iodide is added to the first reconstituted solution, allowed to stand at 2 ℃ to 8 ℃, and centrifuged to provide a third supernatant.
In certain embodiments, iodide is added to the first reconstituted solution such that the concentration of iodide is between 0.5% and 2.0% (w/v).
In certain embodiments, iodide is added to the first reconstituted solution such that the concentration of iodide is from 1.0% to 2.0% (w/v).
In certain embodiments, iodide is added to the first reconstituted solution such that the concentration of iodide is 0.5% to 1.0% (w/v), allowed to stand at 2 ℃ to 8 ℃, and centrifuged to obtain a third supernatant.
In certain embodiments, illustrative examples of iodides that can be used in the present disclosure include, but are not limited to, sodium iodide (NaI) and potassium iodide (KI).
In certain embodiments, the third supernatant is filtered and then freeze-dried to provide the pneumococcal capsular polysaccharide.
In certain embodiments, the third supernatant is ultrafiltered and then freeze dried to provide pneumococcal capsular polysaccharide.
In certain embodiments, the third supernatant is ultrafiltered using a membrane having a pore size of 100KD to 300KD to obtain an ultrafiltration concentrate.
In certain embodiments, illustrative examples of membranes that can be used for ultrafiltration of the third supernatant of the present disclosure include, but are not limited to, ultrafiltration membranes.
In certain embodiments, liquids that can be used for ultrafiltration of the third supernatant of the present disclosure include, but are not limited to, water for injection.
In certain embodiments, the volume of water for injection that can be used for ultrafiltration of the third supernatant of the present disclosure is 15 to 20 times the volume of the supernatant.
In certain embodiments, the methods of the present disclosure do not use ethanol.
In certain embodiments, the methods of the present disclosure do not use toxic and harmful agents such as phenol, acetone, ethanol, and the like, thereby reducing the risk to human health and the environment.
In certain embodiments, the methods of the present disclosure do not use a protease.
In certain embodiments, the methods of the present disclosure do not use nucleases.
In certain embodiments, the methods of the present disclosure do not use expensive exogenous macromolecular substances such as nucleases, proteases, etc., thereby effectively reducing manufacturing costs.
In certain embodiments, the methods of the present disclosure result in polysaccharide preparations that are superior to pharmacopeia standards without the use of nuclease, protease, or ethanol precipitation steps, both avoiding the use of large amounts of organic solvents and simplifying the process flow.
In certain embodiments, CTAB remains less than or equal to 5 μ g/g pneumococcal capsular polysaccharide using the methods of the present disclosure.
In another aspect, the present disclosure relates to pneumococcal capsular polysaccharides prepared by a process comprising the steps of:
providing a pneumococcal fermentation broth;
separating the pneumococcal fermentation liquor to obtain a first supernatant;
filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate;
adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant;
diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate;
dissolving the first precipitate to obtain a first redissolution;
adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide;
wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
In yet another aspect, the present disclosure relates to a pneumococcal vaccine comprising a pneumococcal capsular polysaccharide prepared by a process comprising the steps of:
providing a pneumococcal fermentation broth;
separating the pneumococcal fermentation liquor to obtain a first supernatant;
filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate;
adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant;
diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate;
dissolving the first precipitate to obtain a first redissolution;
adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide;
wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that the concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L.
In certain embodiments, the pneumococcal polysaccharide vaccine is a 23-valent pneumococcal polysaccharide vaccine.
In certain embodiments, the pneumococcal polysaccharide vaccine comprises pneumococcal serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F.
In certain embodiments, the pneumococcal polysaccharide vaccine is a 13-valent pneumococcal polysaccharide conjugate vaccine.
In certain embodiments, the pneumococcal polysaccharide vaccine comprises pneumococcal serotypes 4, 5, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 23F.
In certain embodiments, the pneumococcal polysaccharide vaccine is a 15-valent pneumococcal polysaccharide conjugate vaccine.
In certain embodiments, the pneumococcal polysaccharide vaccine comprises pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, 23F.
In certain embodiments, the pneumococcal polysaccharide vaccine is a 20-valent pneumococcal polysaccharide conjugate vaccine.
In certain embodiments, the pneumococcal polysaccharide vaccine comprises pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F.
Hereinafter, the present disclosure will be explained in detail by the following examples in order to better understand various aspects of the present application and advantages thereof. It should be understood, however, that the following examples are not limiting and are merely illustrative of certain embodiments of the present disclosure.
Examples
Material and equipment sources:
the pneumococcus is purchased from China medical bacteria preservation management center of China food and drug testing research institute.
Ultrafiltration membrane and clamp: 0.1 square meter (available from pall corporation, usa).
A centrifuge: sorvall LYNX 6000 (from Thermo).
Cetyl trimethylammonium bromide (CTAB) available from VWR corporation, usa; the rest of the chemical reagents are purchased from chemical reagents of national drug group, ltd.
Example 1
Pneumococcal type 1 capsular polysaccharide
1. Purification of pneumococcal type 1 capsular polysaccharide
1) Sterilizing type 1 pneumococcus fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.7mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 2.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 1.5 times of the volume of the supernatant to ensure that the concentration of sodium chloride is 0.5mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring uniformly, standing for 2 hours at 4 ℃, forming association precipitation by CTAB and iodide ions, then centrifuging for 60min at 12000g, and collecting the supernatant solution of the polysaccharide;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrated solution, wherein the volume of water for injection used for ultrafiltration is 15 times of the volume of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying is finished.
2. Assay for pneumococcal type 1 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the detection of the nucleic acid content is carried out according to the three parts (generally 0401) of the pharmacopoeia of the people's republic of China 2020 edition; the total nitrogen content is detected according to the three parts of the pharmacopoeia of the people's republic of China 2020 edition (general regulation 0704); the phosphorus content is detected according to the three parts of the 2020 edition pharmacopoeia of the people's republic of China (general regulations 3103); the content of uronic acid is determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0731); the content of O-acetyl is detected according to the three parts (general rule 3117) of the pharmacopoeia of the people's republic of China 2020 edition; the molecular size of the capsular polysaccharide is measured according to the first method in the third 3.1.2.10 of the pharmacopoeia of the people's republic of China 2020 edition. The relevant assay results are shown in table 1.
TABLE 1 recovery and quality control index for three batches of pneumococcal type 1 capsular polysaccharide
Example 2
Pneumococcal type 3 capsular polysaccharide
1. Purification of pneumococcal type 3 capsular polysaccharide
1) Sterilizing type 3 pneumococcal fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 3.5; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with 100 KD-pore membrane to obtain ultrafiltration concentrate, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.8mol/L, and the volume of the NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 2.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 3 times of the volume of the supernatant so that the concentration of sodium chloride is 0.27mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.5% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrated solution, wherein the volume of water for injection used for ultrafiltration is 15 times of the volume of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 3 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the detection of the nucleic acid content is carried out according to the three parts (generally 0401) of the pharmacopoeia of the people's republic of China 2020 edition; the total nitrogen content is detected according to the three parts of the pharmacopoeia of the people's republic of China 2020 edition (general regulation 0704); the phosphorus content is detected according to the three parts of the 2020 edition pharmacopoeia of the people's republic of China (general regulations 3103); the detection of the content of uronic acid is carried out according to the third division of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 0731); the molecular size of the capsular polysaccharide is determined according to the first method in the third section 3.1.2.10 of pharmacopoeia of the people's republic of China 2020. The relevant assay results are shown in table 2.
TABLE 2 recovery and quality control index for three batches of pneumococcal type 3 capsular polysaccharide
Example 3
Pneumococcal type 5 capsular polysaccharide
1. Purification of pneumococcal type 5 capsular polysaccharide
1) Sterilizing a type 5 pneumococcal fermentation liquid, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquid to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.6mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 1.5% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 2.5 times of the volume of the supernatant to ensure that the concentration of sodium chloride is 0.24mol/L, then adding CTAB again according to the concentration of 1.0 percent (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying is finished.
2. Assay for pneumococcal type 5 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the detection of the nucleic acid content is carried out according to three parts (general rule 0401) of the 2020 edition of pharmacopoeia of the people's republic of China; the total nitrogen content is detected according to the three parts of the pharmacopoeia of the people's republic of China 2020 edition (general regulation 0704); the phosphorus content is detected according to the third part of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 3103); the detection of the content of uronic acid is carried out according to the third division of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 0731); the content of hexosamine is detected according to the three parts (general rule 0401) of the 2020 edition pharmacopoeia of the people's republic of China; the molecular size of the capsular polysaccharide is determined according to the first method in the third section 3.1.2.10 of pharmacopoeia of the people's republic of China 2020. The relevant assay results are shown in table 3.
TABLE 3 recovery and quality control indexes of three batches of pneumococcal type 5 capsular polysaccharide
Example 4
Pneumococcal type 8 capsular polysaccharide
1. Purification of pneumococcal type 8 capsular polysaccharide
1) Sterilizing type 8 pneumococcal fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.9mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 2.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 3 times of the volume of the supernatant so that the concentration of sodium chloride is 0.3mol/L, then adding CTAB again according to the concentration of 1.0% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 2.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrated solution, wherein the volume of water for injection used for ultrafiltration is 15 times of the volume of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 8 capsular polysaccharide
Wherein the content of the capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 0731); the detection of the nucleic acid content is carried out according to the three parts (generally 0401) of the pharmacopoeia of the people's republic of China 2020 edition; the total nitrogen content is detected according to the three parts of the pharmacopoeia of the people's republic of China 2020 edition (general regulation 0704); the phosphorus content is detected according to the three parts of the 2020 edition pharmacopoeia of the people's republic of China (general regulations 3103); the content of uronic acid is determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0731); the molecular size of the capsular polysaccharide is measured according to the first method in the third 3.1.2.10 of the pharmacopoeia of the people's republic of China 2020 edition. The relevant assay results are shown in table 4.
TABLE 4 recovery and quality control index for three batches of pneumococcal type 8 capsular polysaccharide
Example 5
12F pneumococcal capsular polysaccharide
1. Purification of pneumococcal 12F capsular polysaccharide
1) Sterilizing 12F type pneumococcal fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 5.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.4mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 1.5% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 2 times of the volume of the supernatant so that the concentration of sodium chloride is 0.2mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying is finished.
2. Assay for pneumococcal type 12F capsular polysaccharide
Wherein the content of the capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the detection of the nucleic acid content is carried out according to the three parts (generally 0401) of the pharmacopoeia of the people's republic of China 2020 edition; the total nitrogen content is detected according to the three parts of the pharmacopoeia of the people's republic of China 2020 edition (general regulation 0704); the phosphorus content is detected according to the three parts of the 2020 edition pharmacopoeia of the people's republic of China (general regulations 3103); the content of hexosamine is detected according to the three parts (general rule 0401) of the 2020 edition pharmacopoeia of the people's republic of China; the molecular size of the capsular polysaccharide is determined according to the first method in the third section 3.1.2.10 of pharmacopoeia of the people's republic of China 2020. The relevant assay results are shown in table 5.
TABLE 5 recovery and quality control index for three batches of pneumococcal type 12F capsular polysaccharide
Comparative example 1
Pneumococcal type 1 capsular polysaccharide
1. Purification of pneumococcal type 1 capsular polysaccharide
1) Sterilizing type 1 pneumococcus fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with 100 KD-pore membrane to obtain ultrafiltration concentrate, wherein the molar concentration of NaCl solution used for ultrafiltration is 1.0mol/L, and the volume of the NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration be 1.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 1.5 times of the volume of the supernatant to ensure that the concentration of sodium chloride is 0.67mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrated solution, wherein the volume of water for injection used for ultrafiltration is 15 times of the volume of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 1 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the nucleic acid content was determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0401). The results of the relevant assays are shown in Table 6.
TABLE 6 recovery and quality control index for three batches of pneumococcal type 1 capsular polysaccharide
Compared with the NaCl solution used in ultrafiltration in the embodiment 1, the molar concentration of the NaCl solution used in ultrafiltration is increased from 0.7mol/L to 1.0mol/L, the recovery rate of the obtained capsular polysaccharide is reduced by 40.0%, the content of protein and nucleic acid impurities is remarkably increased, and the content of the protein impurities exceeds the standard.
Comparative example 2
Pneumococcal type 3 capsular polysaccharide
1. Purification of pneumococcal type 3 capsular polysaccharide
1) Sterilizing type 3 pneumococcus fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 3.5; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 0.5mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 1.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 3 times of the volume of the supernatant so that the concentration of sodium chloride is 0.17mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 3 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the nucleic acid content was determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0401). The relevant assay results are shown in table 7.
TABLE 7 recovery and quality control indexes of three batches of pneumococcal type 3 capsular polysaccharide
Compared with the embodiment 2, the recovery rate of capsular polysaccharide obtained by reducing the molar concentration of NaCl solution used in ultrafiltration from 0.8mol/L to 0.5mol/L is remarkably reduced by 61.4%, the content of protein impurities is remarkably improved, and the content of nucleic acid impurities is remarkably out of limits.
Comparative example 3
Pneumococcal type 5 capsular polysaccharide
1. Purification of pneumococcal type 5 capsular polysaccharide
1) Sterilizing type 5 pneumococcal fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 1.0mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration be 1.5% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 2.5 times of the volume of the supernatant to ensure that the concentration of sodium chloride is 0.4mol/L, then adding CTAB again according to the concentration of 1.0 percent (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the previous step in a NaCl solution with the concentration of 1.0mol/L, and ceaselessly stirring at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying is finished.
2. Assay for pneumococcal type 5 capsular polysaccharide
Wherein the content of the capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the ' pharmacopoeia of the people's republic of China ' 2020 edition (general rule 0731); the nucleic acid content was determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0401). The relevant assay results are shown in table 8.
TABLE 8 recovery and quality control indexes of three batches of pneumococcal type 5 capsular polysaccharide
Compared with the NaCl solution molar concentration used in the ultrafiltration in the embodiment 3, the recovery rate of the capsular polysaccharide obtained by increasing the NaCl solution molar concentration from 0.6mol/L to 1.0mol/L is obviously reduced by 44.0%, and although the protein impurity content is reduced, the nucleic acid impurity content is obviously out of limits.
Comparative example 4
Pneumococcal type 8 capsular polysaccharide
1. Purification of pneumococcal type 8 capsular polysaccharide
1) Sterilizing type 8 pneumococcus fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 4.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 1.0mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 2.0% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 3 times of the volume of the supernatant so that the concentration of sodium chloride is 0.33mol/L, then adding CTAB again according to the concentration of 1.0% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 2.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 8 capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 0731); the nucleic acid content was determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0401). The relevant assay results are shown in table 9.
TABLE 9 recovery and quality control index for three batches of pneumococcal type 8 capsular polysaccharide
Compared with the NaCl solution used in the ultrafiltration in the embodiment 4, the molar concentration of the NaCl solution used in the ultrafiltration is increased from 0.9mol/L to 1.0mol/L, the recovery rate of the capsular polysaccharide is obviously reduced by 24.8%, and the content of protein and nucleic acid impurities is not obviously changed.
Comparative example 5
Pneumococcal type 12F capsular polysaccharide
1. Purification of pneumococcal 12F capsular polysaccharide
1) Sterilizing 12F type pneumococcal fermentation liquor, standing for 12 hours at 4 ℃, and then adding acetic acid to adjust the pH value of the fermentation liquor to 5.0; centrifuging at 12000g for 60min, and collecting supernatant; ultrafiltering the supernatant with membrane with aperture of 100KD to obtain ultrafiltered concentrated solution, wherein the molar concentration of NaCl solution used for ultrafiltration is 1.0mol/L, and the volume of NaCl solution is 5 times of the volume of the supernatant;
2) Adding CTAB into the ultrafiltration concentrated solution obtained in the previous step to make the final concentration of the ultrafiltration concentrated solution be 1.5% (w/v), stirring thoroughly, standing at 4 deg.C for 2 hr, centrifuging at 12000g for 60min, and collecting supernatant;
3) Diluting the supernatant obtained in the last step with water for injection to 2 times of the volume of the supernatant so that the concentration of sodium chloride is 0.5mol/L, then adding CTAB again according to the concentration of 0.5% (w/v), fully stirring, standing for 2 hours at 4 ℃, then centrifuging at 12000g for 10min, and collecting CTAB polysaccharide complex precipitate;
4) Dissolving the CTAB polysaccharide complex obtained in the last step into a NaCl solution with the concentration of 1.0mol/L, and stirring continuously at room temperature until the polysaccharide complex is completely dissolved to obtain a polysaccharide redissolution;
5) Adding sodium iodide into the polysaccharide redissolution obtained in the previous step to make the final concentration of the sodium iodide be 1.0% (w/v), fully stirring, standing at 4 ℃ for 2 hours, precipitating CTAB and iodide ions to form association, centrifuging at 12000g for 60min, and collecting the polysaccharide supernatant solution;
6) Ultrafiltering the polysaccharide supernatant obtained in the previous step with a membrane with a pore size of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is 15 times that of the supernatant;
7) Freeze-drying the ultrafiltration concentrated solution, and collecting polysaccharide after the freeze-drying.
2. Assay for pneumococcal type 12F capsular polysaccharide
Wherein the content of capsular polysaccharide is detected according to a three-part Rate turbidimetric method (3.3.2) in the pharmacopoeia of the people's republic of China 2020 edition; the detection of the protein content is carried out according to the three parts of the 2020 edition of pharmacopoeia of the people's republic of China (general rule 0731); the nucleic acid content was determined according to the three parts of pharmacopoeia of the people's republic of China 2020 edition (general rule 0401). The results of the relevant assays are shown in Table 10.
TABLE 10 recovery and quality control index for three batches of pneumococcal type 12F capsular polysaccharide
Compared with the embodiment 5, the recovery rate of capsular polysaccharide obtained by increasing the molar concentration of NaCl solution used for ultrafiltration from 0.4mol/L to 1.0mol/L is obviously reduced by 72.6%, the content of protein impurities is not obviously changed, but the content of nucleic acid impurities is obviously out of limits.
In the present disclosure, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such variations and modifications are intended to fall within the scope of the appended claims of this disclosure.
Claims (4)
1. A process for preparing a pneumococcal capsular polysaccharide comprising:
providing a pneumococcal fermentation broth;
separating the pneumococcal fermentation liquor to obtain a first supernatant;
filtering the first supernatant by using a first solution containing chloride ions to obtain a first filtrate, wherein the volume of the first solution containing the chloride ions is 5 times of that of the first supernatant;
adding first hexadecyl trimethyl ammonium bromide (CTAB) into the first filtrate, and separating to obtain a second supernatant;
diluting the second supernatant, adding hexacosanyl trimethyl ammonium bromide (CTAB) into the diluted second supernatant, and separating to obtain a first precipitate;
dissolving the first precipitate to obtain a first redissolution;
adding iodide into the first redissolution solution, and separating to obtain a third supernatant; and
filtering and drying the third supernatant to obtain the pneumococcal capsular polysaccharide;
wherein a first cetyltrimethylammonium bromide (CTAB) is added to the first filtrate such that a final concentration of the first cetyltrimethylammonium bromide (CTAB) is 1.0% to 2.5% (w/v); (iii) the concentration of the hexachlorosyltrimethylammonium bromide (CTAB) is 0.25% to 1.5% (w/v); the concentration of the chloride ions in the first solution containing the chloride ions is 0.4mol/L to 0.9mol/L; and the concentration of chloride ions in the diluted second supernatant is 0.2mol/L to 0.5mol/L;
in the method, activated carbon, phenol, acetone, ethanol, nuclease and protease are not used;
in the method, fermentation liquor is sterilized and then stands for 12 hours at 4 ℃, and then acetic acid is added to adjust the pH value of the fermentation liquor to 4.0; then the mixture is separated after 12000g centrifugation for 60 min;
in the method, the step of filtering the first supernatant and the step of filtering the third supernatant are ultrafiltration, and ultrafiltration uses a membrane with the aperture of 100KD for ultrafiltration;
in the method, the first solution containing chloride ions is selected from a sodium chloride solution, a potassium chloride solution, a calcium chloride solution or any mixture thereof;
in the method, the pneumococcal capsular polysaccharide is selected from 1, 3, 5, 8 and 12F.
2. The method of claim 1, wherein the iodide is selected from sodium iodide (NaI), potassium iodide (KI), or any mixture thereof.
3. A pneumococcal capsular polysaccharide prepared by the process of any one of claims 1 to 2.
4. A pneumococcal vaccine comprising the pneumococcal capsular polysaccharide of claim 3.
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