CN117756959B - Preparation method of streptococcus pneumoniae capsular polysaccharide - Google Patents
Preparation method of streptococcus pneumoniae capsular polysaccharide Download PDFInfo
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Abstract
The invention relates to the technical field of biological products, in particular to a preparation method of streptococcus pneumoniae capsular polysaccharide. The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: treating fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting supernatant, purifying the supernatant, and collecting capsular polysaccharide. The beta-propiolactone is used for treating the streptococcus pneumoniae fermentation culture, so that impurity residues such as protein and the like are reduced, and the yield of polysaccharide is higher; moreover, the beta-propiolactone is easy to hydrolyze, and the hydrolysate is nontoxic and harmless, and has higher safety. The quality control indexes of the streptococcus pneumoniae capsular polysaccharide prepared by the method meet the requirements, and the streptococcus pneumoniae capsular polysaccharide can be used for preparing streptococcus pneumoniae capsular polysaccharide vaccines and polysaccharide conjugate vaccines.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a preparation method of streptococcus pneumoniae capsular polysaccharide.
Background
Streptococcus pneumoniae (Streptococcus pneumoniae) is a gram-positive coccus with a capsule, which is the main causative agent of diseases such as human pneumonia, meningitis, otitis media and bacteremia. The diseases caused by streptococcus pneumoniae have higher morbidity and mortality, and the main susceptible population is infants under 5 years old. With the increasing development of Streptococcus pneumoniae resistance, protection against infection by vaccination is increasingly important. The capsular polysaccharide is a key virulence factor of streptococcus pneumoniae, and after the streptococcus pneumoniae is fermented and cultured, the polysaccharide or polysaccharide combined vaccine prepared by extraction, separation, purification and combination can effectively induce human immune response, thereby preventing infection and morbidity of the streptococcus pneumoniae.
Currently, a 23-valent streptococcus pneumoniae polysaccharide vaccine, a 13-valent streptococcus pneumoniae polysaccharide conjugate vaccine and a 20-valent streptococcus pneumoniae polysaccharide conjugate vaccine are commonly used clinically.
The main component of the 23-valent streptococcus pneumoniae polysaccharide vaccine is a plurality of capsular polysaccharide antigens of streptococcus pneumoniae, and the polysaccharide is a T cell independent antigen, so that mature B lymphocytes can be stimulated, but T lymphocytes cannot be stimulated, and the infant immune function under 2 years old is imperfect, and the polysaccharide vaccine can only be used for immunization of people over 2 years old because of poor reaction to the T cell independent antigen.
The combined vaccine of the streptococcus pneumoniae polysaccharide with the valence of 13 and the valence of 20 is also a vaccine taking capsular polysaccharide as a protective antigen, the combined vaccine utilizes a combination technology to combine bacterial capsular polysaccharide with specific carrier protein, the bacterial capsular polysaccharide becomes T cell dependent antigen after combination, immune memory can be formed after inoculation, the immune duration is long, and the combined vaccine is suitable for people of any age group, particularly has good protective effect on infants under 2 years old, and can be applied to the preventive immunity of children or old people.
Currently, in the production of Streptococcus pneumoniae capsular polysaccharides, sodium Deoxycholate (DOC) solution is added to the fermentation broth to lyse the cells and thereby release the capsular polysaccharides in the cells (CN 107835821A, and Ren Keming, bai Guijie, zhang Lizhi, etc.. The establishment of a novel method for purifying Streptococcus pneumoniae capsular polysaccharides [ J ]. J. Chinese biologies, 2020,33 (10): 1181-1185.DOI:10.13200/J. Cnki. Cjb.003178). Sodium deoxycholate is a bile salt, and can quickly activate autolytic enzyme and accelerate self-dissolution of bacteria such as streptococcus pneumoniae and the like. The final stage of fermentation of streptococcus pneumoniae is added with a sodium deoxycholate solution with the final concentration of 0.1%, and bacterial thalli can be lysed, so that capsular polysaccharide on the cell wall surface of the streptococcus pneumoniae is released. However, in this process, not only the capsular polysaccharide attached to the cell wall is released in large amounts, but also the intracellular product of the cell is released into the polysaccharide solution, which makes the subsequent purification very difficult and increases the content of residual impurities (proteins, nucleic acids, etc.). Furthermore, sodium deoxycholate is an animal-derived substance, and may contain exogenous factors (such as bovine-derived viruses) or other sensitizers, which in turn tend to cause stronger side effects or exceed sensitization. In addition, a certain amount of sodium deoxycholate has a damaging effect on gastric mucosal cells, nasal mucosal cells and pancreatic acinar cells. Therefore, if sodium deoxycholate remains in the vaccine preparation, there is also a risk of causing damage to human cells after it is injected into the human body. The current solution is to ensure the safety of vaccine use by establishing a quality control method (such as CN 102830185B) and a standard. There is still a need to develop better solutions capable of reducing, even avoiding, the use of sodium deoxycholate.
In addition, with the increase of the price of the streptococcus pneumoniae vaccine, the residual protein and nucleic acid impurities of the refined polysaccharide in the preparation are accumulated, and a new challenge is brought to the safety and quality control of the streptococcus pneumoniae vaccine. Therefore, to further reduce the total amount of residual proteins and residual nucleic acids in streptococcus pneumoniae polysaccharide vaccines and streptococcus pneumoniae polysaccharide conjugate vaccines, it is desirable to reduce the residual protein and nucleic acid content in individual types of purified polysaccharides.
Disclosure of Invention
The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide.
In the existing production method of streptococcus pneumoniae capsular polysaccharide, sodium deoxycholate is used as a cracking agent for cracking thalli to release capsular polysaccharide, which plays a very important role in extracting capsular polysaccharide, and no substitute reagent can be used for preparing streptococcus pneumoniae capsular polysaccharide at present. The invention surprisingly discovers that beta-propiolactone (BPL) has obvious effect of promoting streptococcus pneumoniae to release capsular polysaccharide, can be used for treating streptococcus pneumoniae to extract capsular polysaccharide, and is different from deoxycholate sodium, the beta-propiolactone can not crack thalli, thereby obviously reducing release of impurities such as protein, nucleic acid and the like in thalli, being beneficial to reducing impurity content and removal difficulty in the purification process of capsular polysaccharide, and the beta-propiolactone is easy to hydrolyze, and hydrolysate is nontoxic and harmless, so that residue or health risk in capsular polysaccharide products can not be caused. Beta-propiolactone is currently used as a virus inactivating agent for preparing virus vaccines, and has not been reported so far regarding the function of promoting streptococcus pneumoniae to release capsular polysaccharide and reducing release of intracellular proteins, nucleic acids and other impurities.
Specifically, the invention provides the following technical scheme:
The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: treating fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting supernatant, purifying the supernatant, and collecting capsular polysaccharide.
In the method, beta-propiolactone is used for replacing sodium deoxycholate to kill streptococcus pneumoniae and release polysaccharide, so that the problems of health risk or side effect caused by sodium deoxycholate residues are solved, the content of protein and other impurities in extracted capsular polysaccharide is obviously reduced, the content of protein and other impurities in purified polysaccharide is obviously reduced (especially for types with high protein content in streptococcus pneumoniae capsular polysaccharide such as 5, 10A, 14, 7F, 15B, etc., the protein residue in capsular polysaccharide prepared by beta-propiolactone treatment thallus is obviously reduced, the impurity removing effect is excellent), the quality is obviously better than that of purified polysaccharide obtained by purifying after splitting thallus by sodium deoxycholate, the recovery rate of polysaccharide is higher, and the recovery rate is obviously higher than that of streptococcus pneumoniae fermentation culture treated by formaldehyde.
In the above method, the fermentation culture is a culture (e.g., fermentation broth) comprising Streptococcus pneumoniae obtained by fermentation culture of Streptococcus pneumoniae.
For the final concentration of beta-propiolactone in the treatment system, it may be added in the amounts usual in the art.
Preferably, the final concentration of the beta-propiolactone in the treatment system is not less than 0.01%.
Exemplary final beta-propiolactone concentrations may be 0.01%, 0.025%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc.
In some embodiments of the invention, the final concentration of the beta-propiolactone in the treatment system is not less than 0.05%.
In some embodiments of the invention, the final concentration of the beta-propiolactone in the treatment system is from 0.05 to 1%. Preferably 0.05-0.5%.
Preferably, the temperature of the treatment is 2-8 ℃.
Preferably, the treatment time is 8-16 hours.
Preferably, the treatment is low temperature incubation after uniform mixing.
In the above-described production method, sodium deoxycholate is preferably not used.
Preferably, the treatment is performed by adding beta-propiolactone (BPL) at the late stage of logarithmic growth of the streptococcus pneumoniae fermentation culture. After adding beta-propiolactone (BPL), stirring and mixing uniformly, and then incubating at low temperature.
In the present invention, the separation of the supernatant may be performed by any solid-liquid separation method, for example, the supernatant may be separated by centrifugation.
It will be appreciated by those skilled in the art that the preparation of the capsular polysaccharide of Streptococcus pneumoniae is divided into a stage of extracting the capsular polysaccharide from the bacterial cells (i.e. separating the capsular polysaccharide from the bacterial cells, first stage) and a purification stage of the capsular polysaccharide. The beta-propiolactone acts as a reagent for promoting the thalli to release the capsular polysaccharide in the first stage, and experiments prove that the beta-propiolactone is used for treating the streptococcus pneumoniae fermentation culture, so that the capsular polysaccharide can be effectively released, and the content of protein and nucleic acid in the capsular polysaccharide extracting solution (supernatant collected after treatment) is obviously reduced compared with that of sodium deoxycholate. Therefore, the present invention is not particularly limited in nature with respect to the subsequent purification method of capsular polysaccharide, as long as impurities such as proteins, nucleic acids, etc. can be effectively removed, so that purification methods (for example, methods for purifying Streptococcus pneumoniae capsular polysaccharides known in the art) in which the purity of the resulting capsular polysaccharide is satisfactory can be used.
Purification as described above includes protein removal and nucleic acid removal.
The above purification comprises one or more selected from ultrafiltration, acid precipitation, salt precipitation, organic solvent precipitation, and chromatography.
The traditional streptococcus pneumoniae capsular polysaccharide purification process generally adopts ethanol fractional precipitation to obtain crude polysaccharide, and then uses phenol extraction, activated carbon adsorption or chromatography and other methods to obtain refined polysaccharide. Although qualified streptococcus pneumoniae capsular polysaccharides can be obtained by using the methods, the method has the defects and limitations that the steps are more, the time is long, and phenol is taken as a reagent with extremely strong corrosiveness, so that a large amount of phenol is seriously harmful to human bodies and the environment.
Preferably, the purification according to the present invention does not use toxic and harmful reagents, including one or more of phenol, acetone.
Preferably, the purification according to the present invention does not use exogenous macromolecular substances, including one or more of nucleases, proteases.
The ultrafiltration is preferably performed using an ultrafiltration membrane having a pore size of 100 to 300 KD a.
The acid precipitation is preferably carried out at a pH of 2.5 to 4.5.
The above salt precipitation is preferably carried out using a precipitant comprising a calcium salt.
The precipitant used in the above-mentioned organic solvent precipitation may be an organic solvent such as a lower alcohol (e.g., ethanol, methanol) or the like.
As an example of a purification method, the purification includes: ultrafiltering the supernatant to obtain a first ultrafiltrate concentrate; performing first precipitation treatment on the first ultrafiltration concentrated solution, and separating supernatant to obtain first supernatant; performing second precipitation treatment on the first supernatant, and separating the supernatant to obtain a second supernatant;
wherein the first precipitation treatment is carried out under the condition that the pH value is 2.8-4.2;
The precipitant used in the second precipitation treatment includes a lower alcohol.
In the purification method, the supernatant of the streptococcus pneumoniae fermentation culture treated by beta-propiolactone is ultrafiltered to remove small molecule residues, and then subjected to a first precipitation treatment (acid precipitation) and a second precipitation treatment (alcohol precipitation) to remove impurities such as protein, nucleic acid and the like, and finally subjected to ultrafiltration concentration and drying to obtain refined polysaccharide.
Preferably, the first precipitation treatment is to stand at 2-8 ℃ for not less than 1h. In the present invention, the first precipitate may be simply referred to as acid precipitate.
In some embodiments of the invention, the first ultrafiltration concentrate may be subjected to constant volume displacement, the displacement solution being water for injection, the volume of water for injection being 4-6 times the volume of concentrate.
In some embodiments of the invention, the first ultrafiltration concentrate is added with an acid solution to adjust the pH to 2.8-4.2, and after being well stirred, is left to stand at 2-8 ℃ for not less than 1 hour, and then the first supernatant is collected by centrifugation.
In other embodiments of the present invention, the first ultrafiltration concentrate is subjected to constant volume displacement, then an acid solution is added to adjust the pH to 2.8-4.2, and after sufficient stirring, the first ultrafiltration concentrate is left to stand at 2-8 ℃ for not less than 1 hour, and then the first supernatant is collected by centrifugation.
Preferably, the precipitants used in the second precipitation treatment include phosphate buffer salts, sodium salts, calcium salts, and lower alcohols.
Wherein the phosphate buffer salt comprises disodium hydrogen phosphate and/or sodium dihydrogen phosphate.
The sodium salt comprises sodium chloride and/or sodium acetate.
The calcium salt comprises calcium chloride.
The lower alcohol includes ethanol.
Preferably, in the second precipitation treatment, the concentration of disodium hydrogen phosphate is 8-12mM, the concentration of sodium dihydrogen phosphate is 8-12mM, the concentration of sodium chloride is 0.1-2.5M, the concentration of sodium acetate is 0.3-1.2M, the concentration of calcium chloride is 0.1-0.35M, and the volume ratio of ethanol to feed liquid is 20% -30%. This step may be referred to herein simply as ethanol precipitation.
In some embodiments, the ethanol may be provided from 50%, 60%, 70%, 80%, 90% ethanol solution or absolute ethanol, preferably absolute ethanol is used.
Preferably, the second precipitation treatment is carried out at a pH of 5.3 to 5.5.
Preferably, the second precipitation treatment is a standing treatment. The treatment time is preferably not less than 3 hours.
Preferably, the pH of the first supernatant is adjusted to 6.8-7.2 with alkali liquor, phosphate buffer salt, sodium salt and calcium salt are added, then the pH is adjusted to 5.3-5.5 with acid liquor, and then lower alcohol is added for the second precipitation treatment.
The pH adjustment to acidity can be performed using acid solutions, including, but not limited to, glacial acetic acid, hydrochloric acid, phosphoric acid, etc.; glacial acetic acid is preferred.
The pH adjustment to alkaline can be performed by alkali lye, including but not limited to sodium hydroxide, potassium hydroxide and the like; sodium hydroxide is preferred.
Preferably, the ultrafiltration is performed by ultrafiltration concentration by using a membrane bag with the pore diameter of 100KD to obtain a first ultrafiltration concentrated solution.
Preferably, the purification further comprises: and ultrafiltering the second supernatant to obtain a second ultrafiltration concentrated solution.
Preferably, the second supernatant is ultrafiltered using a membrane having a pore size of 100-300KD to obtain a second ultrafiltered concentrate.
The volume of water for injection used for ultrafiltration may be 5 times, 6 times, 7 times or 8 times or more, preferably 8 times or more, the volume of the second supernatant.
As an example of another purification method, the purification includes: sequentially performing first ultrafiltration treatment, precipitation treatment and second ultrafiltration treatment on the supernatant;
Wherein the precipitating agent used in the precipitating treatment comprises calcium salt, and the precipitating treatment is carried out under the condition that the pH value is 2.4-3.6.
In the research and development process of the streptococcus pneumoniae capsular polysaccharide preparation method, a simple and rapid streptococcus pneumoniae polysaccharide preparation method is discovered accidentally, the method can prepare high-quality refined polysaccharide by only carrying out one-step precipitation (one-step calcium salt acid precipitation) under an acidic condition and matching with a conventional ultrafiltration step, and various indexes of the prepared refined polysaccharide are higher than the Chinese pharmacopoeia standard, so that the material and time cost is obviously reduced, and the purification efficiency is improved. The use of calcium salt as precipitant and the precipitation treatment (calcium salt acid precipitation) under acidic condition with pH of 2.4-3.6 are key to the above preparation method. The method is matched with the calcium salt acid precipitation, and ultrafiltration treatments are respectively arranged before and after the calcium salt acid precipitation, wherein the primary effect of the first ultrafiltration treatment is to remove small molecular impurities in supernatant fluid after fermentation culture sterilization treatment, so that the calcium salt acid precipitation is more beneficial to better play a role in removing impurities, the impurity removal efficiency is improved, and the primary effect of the second ultrafiltration treatment is to concentrate polysaccharide solution and wash and filter the polysaccharide solution, so as to further remove the small molecular impurities and salt ions in the polysaccharide solution.
Preferably, the calcium salt is calcium chloride.
In the precipitation treatment, the concentration of the calcium salt is 80-200mmol/L.
The precipitation treatment is to collect the supernatant after standing for 1-5 hours at 2-8 ℃ to obtain the first supernatant.
Preferably, the purification further comprises adjusting the pH of the first supernatant obtained by the precipitation treatment to 6.8-7.5, collecting the supernatant to obtain a second supernatant, and subjecting the second supernatant to a second ultrafiltration treatment.
Wherein the first ultrafiltration treatment is performed using an ultrafiltration membrane having a pore size of 100-150KD (preferably 100 KD). In some embodiments of the present invention, the first ultrafiltration treatment is an isovolumetric ultrafiltration after concentration 3-5 times using a membrane package with a pore size of 100 KD. Preferably, the volume of purified water used for ultrafiltration is 4-6 times the volume of the concentrated solution. The second ultrafiltration treatment is performed using an ultrafiltration membrane having a pore size of 100-150 KD. In some embodiments of the invention, the second ultrafiltration treatment uses 8-15 times the volume of the supernatant fluid as the volume of the water for injection used for the wash filtration.
The pH adjustment to acidity can be performed using acid solutions, including, but not limited to, glacial acetic acid, hydrochloric acid, phosphoric acid, etc.; phosphoric acid is preferred.
The pH adjustment to alkaline can be performed by alkali lye, including but not limited to sodium hydroxide, potassium hydroxide and the like; sodium hydroxide is preferred.
Experiments prove that the different purification methods can obtain better refining effect of streptococcus pneumoniae capsular polysaccharide by matching beta-propiolactone to treat streptococcus pneumoniae fermentation cultures, the content of impurities such as protein in the prepared streptococcus pneumoniae capsular polysaccharide is obviously reduced compared with DOC treatment, and the quality control indexes such as specific group content and the like are in accordance with the requirements.
The preparation method further comprises the following steps: and drying the second ultrafiltration concentrated solution.
Preferably, the drying process is a freeze-drying process.
The preparation method of streptococcus pneumoniae capsular polysaccharide in the invention can be used for different types of streptococcus pneumoniae, including but not limited to: 1. types 2, 3,4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.
In some embodiments of the invention, the streptococcus pneumoniae is one or more of types 5, 10A, 14, 7F, 15B.
The invention also provides the streptococcus pneumoniae capsular polysaccharide prepared by the preparation method of the streptococcus pneumoniae capsular polysaccharide.
Preferably, the streptococcus pneumoniae capsular polysaccharide does not contain deoxycholate.
Further preferably, the streptococcus pneumoniae capsular polysaccharide does not contain sodium deoxycholate.
The invention provides a preparation method of the streptococcus pneumoniae capsular polysaccharide or application of the streptococcus pneumoniae capsular polysaccharide in preparation of streptococcus pneumoniae capsular polysaccharide-containing products.
Preferably, the streptococcus pneumoniae capsular polysaccharide product is a vaccine.
The vaccine comprises streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
The invention provides a streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine, which comprises the streptococcus pneumoniae capsular polysaccharide.
The streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine may be a monovalent or multivalent vaccine.
Wherein the multivalent vaccine comprises capsular polysaccharides of at least two types of streptococcus pneumoniae types 1,2,3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F.
Preferably, the multivalent streptococcus pneumoniae capsular polysaccharide vaccine or capsular polysaccharide conjugate vaccine comprises at least one type of capsular polysaccharide from streptococcus pneumoniae types 5, 7F, 10A, 14, 15B.
Specifically, the streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine can be 23-valent streptococcus pneumoniae polysaccharide vaccine, 13-valent streptococcus pneumoniae polysaccharide conjugate vaccine, 20-valent streptococcus pneumoniae polysaccharide conjugate vaccine and 24-valent streptococcus pneumoniae polysaccharide conjugate vaccine.
The 23-valent streptococcus pneumoniae polysaccharide vaccine contains capsular polysaccharides of 1,2, 3,4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F streptococcus pneumoniae serotypes.
The 13-valent streptococcus pneumoniae polysaccharide conjugate vaccine comprises capsular polysaccharides of 1, 3,4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F streptococcus pneumoniae serotypes.
The 20-valent streptococcus pneumoniae polysaccharide conjugate vaccine comprises capsular polysaccharides of 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 19F, 22F, 23F, 33F streptococcus pneumoniae serotypes.
The 24-valent streptococcus pneumoniae polysaccharide conjugate vaccine comprises capsular polysaccharides of 1,2,3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F streptococcus pneumoniae serotypes.
The invention also provides a preparation method of the streptococcus pneumoniae capsular polysaccharide vaccine or the streptococcus pneumoniae capsular polysaccharide conjugate vaccine, which comprises the following steps: the streptococcus pneumoniae capsular polysaccharide is prepared by the preparation method of the streptococcus pneumoniae capsular polysaccharide, and the streptococcus pneumoniae capsular polysaccharide is used as an immunogen to prepare a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
The beneficial effects of the invention at least comprise: the beta-propiolactone is used for treating the streptococcus pneumoniae fermentation culture, so that the effects of inactivating thalli and effectively releasing capsular polysaccharide can be achieved under the condition of not cracking thalli, and a large amount of release of intracellular proteins, nucleic acids and other impurities after the thalli are cracked is avoided, thereby reducing the difficulty of subsequent purification, reducing the residue of the impurities such as proteins and the like, and simultaneously having higher polysaccharide yield; moreover, the beta-propiolactone is easy to hydrolyze, and the hydrolysate is nontoxic and harmless, so that the problems of health risk and side effect caused by the residue of the beta-propiolactone in the finished vaccine are avoided, and the safety is higher. The beta-propiolactone is adopted to replace sodium deoxycholate for treating streptococcus pneumoniae, so that the potential health risk of introducing animal-derived substances and sodium deoxycholate is avoided, and the quality control indexes such as the impurity content of protein, nucleic acid and the like and the specific group content in the prepared streptococcus pneumoniae capsular polysaccharide are all in accordance with the requirements, and the method can be used for preparing streptococcus pneumoniae capsular polysaccharide vaccines and polysaccharide conjugate vaccines.
Detailed Description
The invention provides a preparation method of streptococcus pneumoniae capsular polysaccharide, which comprises the following steps: treating fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting supernatant, purifying the supernatant, and collecting capsular polysaccharide.
Specifically, the preparation method of the streptococcus pneumoniae capsular polysaccharide comprises the following steps:
1. Adding beta-propiolactone (BPL) to the streptococcus pneumoniae fermentation broth cultured to the late logarithmic growth phase, stirring and mixing uniformly, incubating at low temperature for 8-16h, centrifuging the treated fermentation broth, and collecting supernatant;
2. and purifying the supernatant to obtain the refined capsular polysaccharide.
For the purification method, as an example, in some embodiments of the invention, the purification comprises the steps of:
(1) Ultrafiltering and concentrating the supernatant with membrane bag with aperture of 100KD to obtain first ultrafiltered concentrated solution, and performing constant volume displacement on the concentrated solution, wherein the displacement solution is water for injection, and the volume of the water for injection is 4-6 times of that of the concentrated solution;
(2) Adding glacial acetic acid into the first ultrafiltration concentrated solution obtained in the step (1) after constant volume displacement to adjust the pH to 2.8-4.2, fully stirring, standing at 2-8 ℃ for not less than 1h, and centrifuging to collect supernatant;
(3) Adjusting the pH of the supernatant obtained in the step (2) to be neutral by using a NaOH solution, adding disodium hydrogen phosphate and sodium dihydrogen phosphate into the feed liquid, adding sodium chloride, sodium acetate and calcium chloride, fully and uniformly stirring, adding glacial acetic acid to adjust the pH to 5.3-5.5, adding absolute ethyl alcohol, standing for not less than 3 hours, and centrifuging to collect the supernatant;
(4) Ultrafiltering the supernatant obtained in the step (3) by using a membrane with the aperture of 100-300 KD to obtain a second ultrafiltrate concentrate, wherein the volume of water for injection used in ultrafiltration is more than 8 times of the volume of the supernatant;
(5) And (3) freeze-drying the second ultrafiltration concentrated solution obtained in the step (4) to obtain streptococcus pneumoniae capsular polysaccharide.
By way of example, in other embodiments of the invention, the purification comprises the steps of:
(1) Ultrafiltering the supernatant collected by beta-propiolactone treatment with a membrane with the aperture of 100KD to obtain a first ultrafiltrate concentrate;
(2) Adding CaCl 2 solution with final concentration of 80-200mmol/L into the first ultrafiltration concentrated solution, regulating pH to 2.4-3.6 with acid liquor, standing at 2-8deg.C for more than 1h, centrifuging to collect supernatant, regulating pH to 7.00-7.50 with alkali liquor, centrifuging again to collect supernatant;
(3) Ultrafiltering the supernatant obtained in the step (2) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate;
(4) And (3) freeze-drying the polysaccharide ultrafiltration concentrated solution obtained in the step (3) to obtain streptococcus pneumoniae capsular polysaccharide.
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The capsular polysaccharide yields in the following examples and comparative examples were calculated as follows:
DOC treatment completely lyses cells, total capsular polysaccharide release is 100%, yield of capsular polysaccharide when BPL or formaldehyde treatment of fermentation broth is used, calculated based on total polysaccharide in DOC treatment broth:
yield of polysaccharide at BPL or formaldehyde treatment = total amount of polysaccharide in BPL or formaldehyde treatment broth/total amount of polysaccharide in DOC treatment broth.
EXAMPLE 1 preparation of Streptococcus pneumoniae capsular polysaccharide (1)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of streptococcus pneumoniae type 5 as a raw material, and the specific method is as follows:
adding BPL to final concentrations of 0.05% (example 1-001), 0.1% (example 1-002) and 0.2% (example 1-003) respectively into fermentation broth of the clostridium pneumoniae in the late stage of logarithmic growth, stirring uniformly, and incubating at 2-8deg.C for 12 h; centrifuging the treated fermentation broth at 8000rpm for 30min, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
Ultrafiltering and concentrating the supernatant with 100KD membrane bag to obtain concentrated solution, constant volume displacement of the concentrated solution to obtain injection water with volume 4-6 times of that of the concentrated solution, and collecting the first ultrafiltered concentrated solution;
adding glacial acetic acid into the first ultrafiltration concentrated solution to adjust the pH to 4.0, fully stirring, standing at 2-8 ℃ for 1h, and centrifugally collecting supernatant, namely the first supernatant;
Adjusting the pH of the first supernatant to 7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 0.15mol/L of concentration, anhydrous sodium acetate to 0.3mol/L of concentration and calcium chloride (dihydrate) to 0.25mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust the pH to 5.3-5.5 after fully stirring uniformly, adding 20% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for 3h at 2-8 ℃ after stirring uniformly, and centrifuging to collect the supernatant, namely the second supernatant;
And (3) carrying out constant volume displacement on the second supernatant by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and carrying out ultrafiltration concentration on the feed liquid to obtain capsular polysaccharide stock solution.
And freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
Comparative example 1
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 5 as a raw material (Streptococcus pneumoniae type 5 fermentation broth cultured to the late logarithmic growth phase of fermentation, as in example 1, was divided into three parts for example 1, comparative example 1 and comparative example 2, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 1 only in that: BPL was replaced with 0.1% doc.
Comparative example 2
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 5 as a raw material (Streptococcus pneumoniae type 5 fermentation broth cultured to the late logarithmic growth phase of fermentation, as in example 1, was divided into three parts for example 1, comparative example 1 and comparative example 2, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 1 only in that: BPL was replaced with 1% formaldehyde.
Experimental example 1
The polysaccharide yields of the preparation methods of example 1 and comparative examples 1 to 2 were examined for the impurity content of the purified capsular polysaccharide, and the like, and the specific values are as follows:
the detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular weight of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of pharmacopoeia 2020 of the people's republic of China.
The results of the relevant assays are shown in tables 1 and 2.
TABLE 1 quality control index of capsular polysaccharide prepared in example 1 and comparative examples 1 and 2
Note that: comparative examples 1 to 001, 002 and 003 were run in parallel and compared with examples 1 to 001, 002 and 003, respectively, and the other comparative examples were analogized.
TABLE 2 polysaccharide content and recovery of fermentation broths treated in example 1 and comparative examples 1 and 2
The results show that the total polysaccharide amount after BPL treatment (example 1) is more than 70% of the total polysaccharide amount after DOC treatment (comparative example 1), and the total polysaccharide amount after formaldehyde treatment (comparative example 2) is only about 40% of the total polysaccharide amount after DOC treatment; compared with DOC treatment, the reduction amplitude of the impurity protein content in the refined polysaccharide obtained by BPL treatment and formaldehyde treatment can reach more than 30%, the quality of the refined polysaccharide is obviously superior to that of the refined polysaccharide obtained by DOC treatment, and the protein content of the refined polysaccharide obtained by BPL treatment is not obviously different from that of the refined polysaccharide obtained by formaldehyde treatment. Therefore, BPL can be used as a streptococcus pneumoniae bactericide to obtain high-quality refined polysaccharide and relatively high polysaccharide recovery rate.
EXAMPLE 2 preparation of Streptococcus pneumoniae capsular polysaccharide (2)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of 10A streptococcus pneumoniae as a raw material, and the specific method is as follows:
adding BPL to final concentration of 0.05% (example 2-001) and 0.1% (example 2-002) and 0.2% (example 2-003) into the 10A streptococcus pneumoniae fermentation broth cultured to the late logarithmic growth phase, stirring uniformly, and incubating at 2-8deg.C for 8 h; centrifuging the treated fermentation broth at 8000rpm for 30min, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
Ultrafiltering and concentrating the supernatant with 100KD membrane bag to obtain concentrated solution, constant volume displacement of the concentrated solution to obtain injection water with volume 4-6 times of that of the concentrated solution, and collecting the first ultrafiltered concentrated solution;
adding glacial acetic acid into the first ultrafiltration concentrated solution to adjust the pH to 4.0, fully stirring, standing at 2-8 ℃ for 1h, and centrifugally collecting supernatant, namely the first supernatant;
Adjusting the pH of the first supernatant to 7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 0.9mol/L of concentration, anhydrous sodium acetate to 1.2mol/L of concentration and calcium chloride (dihydrate) to 0.25mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust the pH to 5.3-5.5 after fully stirring uniformly, adding 30% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for 3h at 2-8 ℃ after stirring uniformly, and centrifuging to collect the supernatant, namely the second supernatant;
And (3) carrying out constant volume displacement on the second supernatant by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and carrying out ultrafiltration concentration on the feed liquid to obtain capsular polysaccharide stock solution.
And freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
Comparative example 3
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 10A as a raw material (the same as the raw material of example 2, a Streptococcus pneumoniae type 5 fermentation broth cultured to a late logarithmic growth phase of fermentation, divided into three parts, used in example 2, comparative example 3 and comparative example 4, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 2 only in that: BPL was replaced with 0.1% doc.
Comparative example 4
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 10A as a raw material (the same as the raw material of example 2, a Streptococcus pneumoniae type 5 fermentation broth cultured to a late logarithmic growth phase of fermentation, divided into three parts, used in example 2, comparative example 3 and comparative example 4, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 2 only in that: BPL was replaced with 1% formaldehyde.
Experimental example 2
The polysaccharide yields of the preparation methods of example 2 and comparative examples 3 to 4 were examined for the impurity content of the purified capsular polysaccharide, and the like, and the specific values are as follows:
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular weight of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of pharmacopoeia 2020 of the people's republic of China.
The results of the relevant assays are shown in tables 3 and 4.
TABLE 3 quality control indicators for capsular polysaccharides prepared in example 2 and comparative examples 3 and 4
TABLE 4 polysaccharide content and recovery of the fermentation broths treated in example 2 and comparative examples 3 and 4
The results show that the total polysaccharide after BPL treatment (example 2) reached about 70% of the total polysaccharide after DOC treatment (comparative example 3), and that the total polysaccharide after formaldehyde treatment (comparative example 4) was only about 45% of the total polysaccharide after DOC treatment; compared with DOC treatment, the reduction amplitude of the impurity protein content of the refined polysaccharide obtained by BPL treatment and formaldehyde treatment can reach more than 72%, the quality of the refined polysaccharide is obviously superior to that of the refined polysaccharide obtained by DOC treatment, and the impurity protein content of the refined polysaccharide obtained by BPL treatment is not obviously inferior to that of the refined polysaccharide obtained by formaldehyde treatment. Therefore, BPL can be used as a streptococcus pneumoniae bactericide to obtain high-quality refined polysaccharide and relatively high polysaccharide recovery rate.
EXAMPLE 3 preparation of Streptococcus pneumoniae capsular polysaccharide (3)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of type 14 streptococcus pneumoniae as a raw material, and the specific method is as follows:
adding BPL to the fermentation broth of Streptococcus pneumoniae 14 (example 3-001 batch), 0.1% (example 3-002 batch), 0.2% (example 3-003 batch) to a final concentration of 0.05% (example 3-001 batch), stirring uniformly, and incubating at 2-8deg.C for 10 h; centrifuging the treated fermentation broth at 8000rpm for 30min, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
Ultrafiltering and concentrating the supernatant with 100KD membrane bag to obtain concentrated solution, constant volume displacement of the concentrated solution to obtain injection water with volume 4-6 times of that of the concentrated solution, and collecting the first ultrafiltered concentrated solution;
adding glacial acetic acid into the first ultrafiltration concentrated solution to adjust the pH to 4.0, fully stirring, standing at 2-8 ℃ for 1h, and centrifugally collecting supernatant, namely the first supernatant;
adjusting the pH of the first supernatant to 7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 2.5mol/L of concentration, anhydrous sodium acetate to 0.9mol/L of concentration and calcium chloride (dihydrate) to 0.35mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust the pH to 5.3-5.5 after fully stirring uniformly, adding 25% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for 3h at 2-8 ℃ after stirring uniformly, and centrifuging to collect the supernatant, namely the second supernatant;
And (3) carrying out constant volume displacement on the second supernatant by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and carrying out ultrafiltration concentration on the feed liquid to obtain capsular polysaccharide stock solution.
And freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
Comparative example 5
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 14 as a raw material (the same as the raw material of example 3, a Streptococcus pneumoniae fermentation broth of type 14 cultured to the late logarithmic growth phase of fermentation was equally divided into three parts for example 3, comparative example 5 and comparative example 6 respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 3 only in that: BPL was replaced with 0.1% doc.
Comparative example 6
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 14 as a raw material (the same as the raw material of example 3, a Streptococcus pneumoniae fermentation broth of type 14 cultured to the late logarithmic growth phase of fermentation was equally divided into three parts for example 3, comparative example 5 and comparative example 6 respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 3 only in that: BPL was replaced with 1% formaldehyde.
Experimental example 3
The polysaccharide yields of the preparation methods of example 3 and comparative examples 5 to 6 were examined for the impurity content of the purified capsular polysaccharide, and the like, and the specific values are as follows:
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular weight of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in tables 5 and 6.
TABLE 5 quality control indicators for capsular polysaccharides prepared in example 3 and comparative examples 5 and 6
TABLE 6 polysaccharide content and recovery of the fermentation broths treated in example 3 and comparative examples 5 and 6
The results show that the total polysaccharide content after BPL treatment (example 3) reaches about 70% of the total polysaccharide content after DOC treatment (comparative example 5), the total polysaccharide content after formaldehyde treatment (comparative example 6) is only about 47% of the total polysaccharide content after DOC treatment, and the protein content of the purified polysaccharide obtained by BPL treatment is not significantly different from the protein content of the purified polysaccharide obtained by formaldehyde treatment, but compared with DOC treatment, the protein content reduction amplitude of the purified polysaccharide obtained by BPL treatment can reach more than 57%, and the quality of the purified polysaccharide obtained by BPL treatment is significantly better than that of the purified polysaccharide obtained by DOC treatment. Therefore, BPL can be used as a streptococcus pneumoniae bactericide to obtain high-quality refined polysaccharide and relatively high polysaccharide recovery rate.
EXAMPLE 4 preparation of Streptococcus pneumoniae capsular polysaccharide (4)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of 7F streptococcus pneumoniae as a raw material, and the specific method is as follows:
Adding BPL to final concentration of 0.05% (example 4-001) and 0.1% (example 4-002) and 0.2% (example 4-003) into the 7F streptococcus pneumoniae fermentation broth cultured to the late logarithmic growth phase, stirring uniformly, and incubating at 2-8deg.C for 16h; centrifuging the treated fermentation broth at 8000rpm for 30min, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
Ultrafiltering and concentrating the supernatant with 100KD membrane bag to obtain concentrated solution, constant volume displacement of the concentrated solution to obtain injection water with volume 4-6 times of that of the concentrated solution, and collecting the first ultrafiltered concentrated solution;
adding glacial acetic acid into the first ultrafiltration concentrated solution to adjust the pH to 4.0, fully stirring, standing at 2-8 ℃ for 1h, and centrifugally collecting supernatant, namely the first supernatant;
Adjusting the pH of the first supernatant to 7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 0.3mol/L of concentration, anhydrous sodium acetate to 1.2mol/L of concentration and calcium chloride (dihydrate) to 0.2mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust the pH to 5.3-5.5 after fully stirring uniformly, adding 25% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for 3h at 2-8 ℃ after stirring uniformly, and centrifuging to collect the supernatant, namely the second supernatant;
And (3) carrying out constant volume displacement on the second supernatant by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and carrying out ultrafiltration concentration on the feed liquid to obtain capsular polysaccharide stock solution.
And freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
Comparative example 7
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 7F as a raw material (the same raw material as in example 4, a fermentation broth of Streptococcus pneumoniae type 7F cultured to a late stage of logarithmic growth of fermentation, divided into three parts, used in example 4, comparative example 7 and comparative example 8, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 4 only in that: BPL was replaced with 0.1% doc.
Comparative example 8
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 7F as a raw material (the same raw material as in example 4, a fermentation broth of Streptococcus pneumoniae type 7F cultured to a late stage of logarithmic growth of fermentation, divided into three parts, used in example 4, comparative example 7 and comparative example 8, respectively) to prepare capsular polysaccharides, and the preparation method of the capsular polysaccharides of Streptococcus pneumoniae used differs from that of example 4 only in that: BPL was replaced with 1% formaldehyde.
Experimental example 4
The polysaccharide yields of the preparation methods of example 4 and comparative examples 7 to 8 and the impurity content of the purified capsular polysaccharide obtained were examined, and the like were specifically as follows:
the detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the content of the methylpentanose is carried out in three parts (general rule 0401) of the pharmacopoeia of the people's republic of China 2020 edition; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of the pharmacopoeia of the people's republic of China 2020. The results of the relevant assays are shown in tables 7 and 8.
TABLE 7 quality control indicators for capsular polysaccharides prepared in example 4 and comparative examples 7 and 8
TABLE 8 polysaccharide content and recovery of the fermentation broths treated in example 4 and comparative examples 7 and 8
The results show that the total polysaccharide after BPL treatment (example 4) reached about 73% of the total polysaccharide after DOC treatment (comparative example 7), and that the total polysaccharide after formaldehyde treatment (comparative example 8) was only about 47% of the total polysaccharide after DOC treatment; the protein content of the refined polysaccharide obtained by BPL treatment is not obviously different from that of the refined polysaccharide obtained by formaldehyde treatment, but compared with DOC treatment, the protein content of the refined polysaccharide obtained by BPL treatment is reduced by more than 57 percent, and the quality of the refined polysaccharide is obviously better than that of the refined polysaccharide obtained by DOC treatment. Therefore, BPL can be used as a streptococcus pneumoniae bactericide to obtain high-quality refined polysaccharide and relatively high polysaccharide recovery rate.
EXAMPLE 5 preparation of Streptococcus pneumoniae capsular polysaccharide (5)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of 15B streptococcus pneumoniae as a raw material, and the specific method is as follows:
adding BPL to final concentration of 0.05% (example 5-001) and 0.1% (example 5-002) and 0.2% (example 5-003) into 15B streptococcus pneumoniae fermentation broth cultured to late logarithmic growth phase, stirring, and incubating at 2-8deg.C for 14 h; centrifuging the treated fermentation broth at 8000rpm for 30 min, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
Ultrafiltering and concentrating the supernatant with 100KD membrane bag to obtain concentrated solution, constant volume displacement of the concentrated solution to obtain injection water with volume 4-6 times of that of the concentrated solution, and collecting the first ultrafiltered concentrated solution;
adding glacial acetic acid into the first ultrafiltration concentrated solution to adjust the pH to 4.0, fully stirring, standing at 2-8 ℃ for 1h, and centrifugally collecting supernatant, namely the first supernatant;
Adjusting the pH of the first supernatant to 7.0 by using a 5M NaOH solution, adding disodium hydrogen phosphate to 10mmol/L of concentration, sodium dihydrogen phosphate (monohydrate) to 10mmol/L of concentration, sodium chloride to 0.15mol/L of concentration, anhydrous sodium acetate to 0.9mol/L of concentration and calcium chloride (dihydrate) to 0.15mol/L of concentration according to the volume of the feed liquid, adding glacial acetic acid to adjust the pH to 5.3-5.5 after fully stirring uniformly, adding 30% (v/v) of absolute ethyl alcohol according to the volume of the feed liquid, standing for 3h at 2-8 ℃ after stirring uniformly, and centrifuging to collect the supernatant, namely the second supernatant;
And (3) carrying out constant volume displacement on the second supernatant by using a membrane bag with the aperture of 100KD, wherein the displacement solution is water for injection, the volume of the water for injection is more than 8 times of that of the supernatant, and carrying out ultrafiltration concentration on the feed liquid to obtain capsular polysaccharide stock solution.
And freeze-drying the capsular polysaccharide solution, and collecting refined polysaccharide after freeze-drying.
Comparative example 9
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 15B as a raw material (the same raw material as in example 5, a fermentation broth of Streptococcus pneumoniae type 15B cultured to a late stage of logarithmic growth of fermentation, divided into three parts, used in example 5, comparative example 9 and comparative example 10, respectively) to prepare capsular polysaccharides, and the preparation method of Streptococcus pneumoniae capsular polysaccharides used differs from that of example 5 only in that: BPL was replaced with 0.1% doc.
Comparative example 10
This comparative example uses a fermentation culture of Streptococcus pneumoniae type 15B as a raw material (the same raw material as in example 5, a fermentation broth of Streptococcus pneumoniae type 15B cultured to a late stage of logarithmic growth of fermentation, divided into three parts, used in example 5, comparative example 9 and comparative example 10, respectively) to prepare capsular polysaccharides, and the preparation method of Streptococcus pneumoniae capsular polysaccharides used differs from that of example 5 only in that: BPL was replaced with 1% formaldehyde.
Experimental example 5
The polysaccharide yields of the preparation methods of example 5 and comparative examples 9 to 10 were examined for the impurity content of the purified capsular polysaccharide, and the like, and the specific values are as follows:
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the hexosamine content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular weight of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in tables 9 and 10.
TABLE 9 quality control indicators for capsular polysaccharides prepared in example 5 and comparative examples 9 and 10
TABLE 10 polysaccharide content and recovery of fermentation broths treated in example 5 and comparative examples 9 and 10
The results show that the total polysaccharide after BPL treatment (example 5) reached about 68% of the total polysaccharide after DOC treatment (comparative example 9), and that the total polysaccharide after formaldehyde treatment (comparative example 10) was only about 49% of the total polysaccharide after DOC treatment; the protein content of the refined polysaccharide obtained by BPL treatment has no obvious difference with that of the refined polysaccharide obtained by formaldehyde treatment, but compared with DOC treatment, the protein content of the refined polysaccharide obtained by BPL treatment can be reduced by more than 62 percent, and the quality of the refined polysaccharide is obviously better than that of the refined polysaccharide obtained by DOC treatment. Therefore, BPL can be used as a streptococcus pneumoniae bactericide to obtain high-quality refined polysaccharide and relatively high polysaccharide recovery rate.
In conclusion, the streptococcus pneumoniae capsular polysaccharide prepared by treating streptococcus pneumoniae with beta-propiolactone has a protein removal rate which is obviously higher than that of DOC treatment and is basically equal to that of formaldehyde treatment, but the release amount of the polysaccharide is obviously higher than that of formaldehyde treatment, and all quality indexes of the prepared capsular polysaccharide meet the requirements of Chinese pharmacopoeia standards.
EXAMPLE 6 preparation of Streptococcus pneumoniae capsular polysaccharide (6)
The embodiment provides a preparation method of streptococcus pneumoniae capsular polysaccharide, and the capsular polysaccharide is prepared by taking a fermentation culture of type 14 streptococcus pneumoniae as a raw material, and the specific method is as follows:
Adding beta-propiolactone to the fermentation broth of streptococcus pneumoniae of 14 type in the late phase of logarithmic growth of fermentation, stirring uniformly, incubating for 12h at 4 ℃, centrifuging for 30 min at 12000g, and collecting supernatant; the supernatant was purified to prepare a purified polysaccharide, the specific purification process was as follows:
(1) Ultrafiltering the supernatant with 100KD membrane to obtain ultrafiltered concentrated solution, concentrating to obtain supernatant with volume 1/5-1/4 of that of the supernatant and purified water volume 5 times the volume of the concentrated solution;
(2) Adding CaCl 2 solution with final concentration of 80mmol/L (14-2308001), 120mmol/L (14-2308002) and 200mmol/L (14-2308003) into the ultrafiltration concentrated solution obtained in the step (1), fully stirring, regulating the pH to 3.00+/-0.1, fully stirring, standing at 2-8 ℃ for 5h, centrifuging at 12000g for 30 min, collecting supernatant, adjusting the pH to 7.00-7.50 by NaOH, centrifuging at 12000g for 30 min again, and collecting supernatant;
(3) Ultrafiltering the supernatant obtained in the step (2) by using a membrane with the aperture of 100KD to obtain polysaccharide ultrafiltration concentrate, wherein the volume of water for injection used in ultrafiltration is more than 10 times of the volume of the supernatant;
(4) And (3) freeze-drying the ultrafiltration concentrated solution obtained in the step (3), and collecting refined polysaccharide after the completion of the freeze-drying.
Experimental example 6
The streptococcus pneumoniae capsular polysaccharide 14 prepared by the preparation method of example 6 was assayed as follows:
The detection of the content of capsular polysaccharide is carried out according to the turbidimetry (3.3.2) in the three parts of 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 (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the nucleic acid content is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the total nitrogen content is carried out according to the three parts (general rule 0704) of the pharmacopoeia 2020 edition of the people's republic of China; the verification of the phosphorus content is carried out according to the three parts (general rule 3103) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the uronic acid content is carried out according to the three parts (general rule 0731) of the pharmacopoeia 2020 edition of the people's republic of China; the detection of the methylpentanose is carried out according to the three parts (general rule 0401) of the pharmacopoeia 2020 edition of the people's republic of China; the determination of the molecular size of the capsular polysaccharide is carried out according to the first method in the three part 3.1.2.10 of the pharmacopoeia 2020 of the people's republic of China. The results of the relevant assays are shown in Table 11.
Table 11 quality control index of Streptococcus pneumoniae capsular polysaccharide
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (12)
1. A method of preparing streptococcus pneumoniae capsular polysaccharides, the method comprising: treating fermentation culture of Streptococcus pneumoniae with beta-propiolactone, separating and collecting supernatant, purifying the supernatant, and collecting capsular polysaccharide;
Wherein the beta-propiolactone is used as an agent for promoting the release of capsular polysaccharide by thalli, and the temperature of the treatment is 2-8 ℃.
2. The method for preparing streptococcus pneumoniae capsular polysaccharides of claim 1 wherein the final concentration of beta-propiolactone in the treatment system is not less than 0.01%.
3. The method of producing streptococcus pneumoniae capsular polysaccharides according to claim 2, wherein the treatment time is 8-16 hours.
4. A method of preparing streptococcus pneumoniae capsular polysaccharide according to any of claims 1-3 wherein the purification comprises protein removal and nucleic acid removal;
And/or the purification comprises one or more selected from ultrafiltration, acid precipitation, salt precipitation, organic solvent precipitation, chromatography.
5. The method of producing streptococcus pneumoniae capsular polysaccharides according to claim 4, wherein the purification comprises: ultrafiltering the supernatant to obtain a first ultrafiltrate concentrate; performing first precipitation treatment on the first ultrafiltration concentrated solution, and separating supernatant to obtain first supernatant; performing second precipitation treatment on the first supernatant, and separating the supernatant to obtain a second supernatant;
wherein the first precipitation treatment is carried out under the condition that the pH value is 2.8-4.2;
The precipitant used in the second precipitation treatment includes a lower alcohol.
6. The method for preparing streptococcus pneumoniae capsular polysaccharides of claim 5, wherein the precipitants used in the second precipitation treatment comprise phosphate buffer, sodium salts, calcium salts and lower alcohols.
7. The method of preparing streptococcus pneumoniae capsular polysaccharides of claim 6 wherein the phosphate buffer salts comprise disodium hydrogen phosphate and/or sodium dihydrogen phosphate;
And/or, the sodium salt comprises sodium chloride and/or sodium acetate;
And/or, the calcium salt comprises calcium chloride;
and/or, the lower alcohol comprises ethanol.
8. The method for preparing streptococcus pneumoniae capsular polysaccharide according to any of claims 5-7, wherein the purification further comprises: and ultrafiltering the second supernatant to obtain a second ultrafiltration concentrated solution.
9. The streptococcus pneumoniae capsular polysaccharide prepared by the method for preparing streptococcus pneumoniae capsular polysaccharide according to any one of claims 1-8.
10. The method for preparing streptococcus pneumoniae capsular polysaccharide according to any one of claims 1-8 or the use of streptococcus pneumoniae capsular polysaccharide according to claim 9 in the preparation of a product comprising streptococcus pneumoniae capsular polysaccharide.
11. A streptococcus pneumoniae capsular polysaccharide vaccine or streptococcus pneumoniae capsular polysaccharide conjugate vaccine, characterized in that the vaccine comprises a streptococcus pneumoniae capsular polysaccharide according to claim 9.
12. A method of preparing a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine, the method comprising: the streptococcus pneumoniae capsular polysaccharide prepared by the preparation method of the streptococcus pneumoniae capsular polysaccharide according to any one of claims 1-8, wherein the streptococcus pneumoniae capsular polysaccharide is used as an immunogen to prepare a streptococcus pneumoniae capsular polysaccharide vaccine or a streptococcus pneumoniae capsular polysaccharide conjugate vaccine.
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