CN116121106A - Streptococcus pneumoniae animal-derived freeze-drying protective agent - Google Patents
Streptococcus pneumoniae animal-derived freeze-drying protective agent Download PDFInfo
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- C12N1/04—Preserving or maintaining viable microorganisms
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Abstract
The invention discloses a streptococcus pneumoniae animal-derived freeze-drying protective agent, and belongs to the field of microbial preservation. The freeze-drying protective agent consists of the following components: 1 to 5 percent of soybean peptone, 1 to 5 percent of trehalose, 4 to 8 percent of mannitol, 0.3 to 1.5 percent of glycerol and the balance of water. The freeze-drying protective agent can better maintain the activity of streptococcus pneumoniae, has the effect equivalent to that of a skimmed milk protective agent or a commercially available animal-source-free freeze-drying protective agent, does not contain animal-source components, and has low cost.
Description
The present application is a divisional application filed with the invention patent application with the application number of 202110776003.4 and the application date of 2021, 07 and 08.
Technical Field
The invention belongs to the field of microbial preservation, and particularly relates to a streptococcus pneumoniae animal-derived freeze-drying protective agent.
Background
The freeze drying is a drying method which comprises the steps of cooling and freezing a water-containing substance into a solid in advance, and then dehydrating the substance at a low temperature by utilizing the sublimation performance of water under the condition of low temperature and reduced pressure so as to achieve the drying purpose. Freeze drying can retain the activity of bacteria to a large extent, and the live bacteria products usually need to be subjected to freeze drying treatment before storage and transportation. However, the various stresses created by the freeze-drying process can cause some damage and even death to some microbial cells, requiring the addition of lyoprotectants to maintain microbial activity as much as possible.
Lyoprotectants can be mainly classified into several classes of complexes, including skim milk, serum, peptone, gelatin, polypeptides, dextrin, methylcellulose, etc., sugars, including sucrose, lactose, maltose, trehalose, etc., alcohols, amino acids, polymers, surfactants, antioxidants, etc., each of which contains various specific substances (Zhang Guanglei, development of viable bacteria preparation lyoprotectants, microbiological immunology, 2015, 8, 43, 4).
Streptococcus pneumoniae is a gram-positive bacterium that causes various diseases such as pneumonia, meningitis, bacteremia, and the like. Preservation of streptococcus pneumoniae is related to the quality of the raw materials of the streptococcus pneumoniae vaccine, and thus streptococcus pneumoniae is important in the production of related vaccines.
The publication No. CN 111849781A discloses a Streptococcus pneumoniae freeze-drying protective agent comprising 10% by weight of skimmed milk powder, 5% by weight of sucrose, 2% by weight of glycerol, 1% by weight of sorbitol, 0.5% by weight of sodium glutamate, 5% by weight of trehalose, 0.1% by weight of vitamin C, 1% by weight of mannitol and 1.5% by weight of defibrinated sheep blood, which can enable the survival rate of the thalli after freeze-drying to be 84%. However, the protectant contains a large proportion of animal-derived components, namely skimmed milk powder and defibrinated sheep blood, which has the disadvantages that: on one hand, the cost is high; on the other hand, pathogens or sensitizers of animal origin are easily mixed in, which brings potential safety hazard to vaccine products.
Disclosure of Invention
The invention aims to solve the problems that: provides a streptococcus pneumoniae animal-derived freeze-drying protective agent.
The technical scheme of the invention is as follows:
a streptococcus pneumoniae animal-derived lyoprotectant comprises the following components:
1 to 5 percent of soybean peptone, 1 to 5 percent of trehalose, 4 to 8 percent of mannitol, 0.3 to 1.5 percent of glycerol and the balance of water.
Further, the protective agent consists of the following components:
1 to 4 percent of soybean peptone, 1 to 4 percent of trehalose, 6 to 7 percent of mannitol and 0.6 to 1.2 percent of glycerol, and the balance of water.
Further, the protective agent consists of the following components:
1.5 to 2.5 percent of soybean peptone, 1.5 to 2.5 percent of trehalose, 6 to 7 percent of mannitol and 0.6 to 0.7 percent of glycerol, and the balance of water.
Further, the protective agent consists of the following components:
2% w/v soytone, 2% w/v trehalose, 6% w/v mannitol and 0.6% v/v glycerol, the balance being water.
A method for freeze-drying streptococcus pneumoniae includes such steps as mixing the freeze-drying protecting agent with the bacterial deposit of streptococcus pneumoniae, preparing bacterial suspension, and freeze-drying.
Further, the serotype of streptococcus pneumoniae is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
The lyophilized powder is a product obtained by mixing the lyoprotectant and bacterial precipitation of streptococcus pneumoniae to prepare bacterial suspension and lyophilizing.
As in the previous lyophilized powder, the serotype of Streptococcus pneumoniae is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
The streptococcus pneumoniae vaccine is prepared by using the freeze-dried powder, fermenting, purifying and carrying out necessary chemical treatment, and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
The freeze-dried protective agent or the freeze-dried powder is used for preparing streptococcus pneumoniae vaccines.
The invention has the beneficial effects that:
1. the components in the animal-source-free freeze-drying protective agent are matched with each other, so that the streptococcus pneumoniae activity can be effectively protected in the freeze-drying process, the effect is equivalent to that of a commercial product, and the cost is less than 1/10 of that of the commercial product.
2. The animal-source-free freeze-drying protective agent can help to maintain the activity of thalli after the streptococcus pneumoniae is freeze-dried, and the activity is hardly reduced when the agent is stored for 1 year at the temperature of 2-8 ℃; the activity decrease rate was slow when stored at 25℃for 32 weeks. While closely-composed protectants (soy peptone replaced with sodium glutamate) have difficulty maintaining the activity of Streptococcus pneumoniae for a long period of time, streptococcus pneumoniae has difficulty surviving stably at 25+ -2deg.C for more than 2 weeks.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: post lyophilization survival of skimmed milk, commercially available animal-derived lyoprotectants (commercially available), and streptococcus pneumoniae serotype 7F under protection of example 1 lyoprotectant (homemade formulation).
Fig. 2: example 1 lyoprotectants and skim milk protectants lyophilized strain acceleration stability (25.+ -. 2 ℃) after lyophilization of streptococcus pneumoniae serotype 18C, respectively.
Fig. 3: 32 week accelerated stability (25.+ -. 2 ℃) of lyophilized species of Streptococcus pneumoniae of 13 serotypes.
Fig. 4: example 1 lyoprotectants were involved in the real-time stability (2-8 ℃) assay after lyophilization of the protected 13 serotypes of streptococcus pneumoniae.
Fig. 5: streptococcus pneumoniae freeze-dried bacterial appearance.
Fig. 6: effects of single-component lyoprotectants on lyophilized survival of streptococcus pneumoniae serotype 7F.
Fig. 7: effect of several combined lyoprotectants on the survival rate of streptococcus pneumoniae serotype 9V lyophilization. Only the lyoprotectant with acceptable appearance and skim milk are shown in the figure.
Fig. 8: lyophilized survival rate of lyophilized Streptococcus pneumoniae serotype 4 in combination of skimmed milk and trehalose + mannitol + glycerol + sodium glutamate.
Fig. 9: after freeze-drying of Streptococcus pneumoniae serotype 18C respectively, skimmed milk, trehalose + mannitol + glycerol + sodium glutamate (comparative example 3) and trehalose + mannitol + glycerol + soytone (example 1), the viable count of the freeze-dried species was varied at 25.+ -. 2 ℃.
Detailed Description
The Streptococcus pneumoniae strains used in this section include 13 serotypes, each as shown in Table 1.
TABLE 1 serotype of strains
| Serotypes | CMCC (China medical Collection center) |
| 1 | 31401 |
| 3 | 31436 |
| 4 | 31446 |
| 5 | 31456 |
| 6A | 31476 |
| 6B | 31489 |
| 7F | 31507 |
| 9V | 31546 |
| 14 | 31601 |
| 18C | 31687 |
| 19A | 31704 |
| 19F | 31692 |
| 23F | 31759 |
The 13 serotype streptococcus pneumoniae strains used in this section were all purchased from the chinese medical bacterial collection center (CMCC).
Among them, the above-mentioned Streptococcus pneumoniae with a bacterial number of 31446 is proved to be commercially available from the China center for medical collection of bacteria (CMCC) in the literature ("study of large-scale fermentation process conditions for Streptococcus pneumoniae by empirical amplification", zhang Xinzhuang et al, microbiological immunology progress, volume 44, 2016, 12, 6, 49 left column, section 1.1, 1.3).
Streptococcus pneumoniae having a bacterial number of 31401 has been demonstrated to be commercially available in the literature ("laboratory study for detection of fungal infections by polymerase chain reaction", chinese medical guidelines, liu Dafang et al, 2018,16 (27), 116-117).
Streptococcus pneumoniae having a bacterial number of 31436 has been demonstrated in the literature ("influence of co-inoculation of various pneumococcal heat shock proteins on the resistance of mice to pneumococci", natal literature, et al, journal of Chinese biologics 2016,29 (03), 237-241) that this strain can be purchased from the China medical collection of bacteria (CMCC).
The streptococcus pneumoniae with the bacterial number 31507 is proved to be commercially available in the patent (application of streptococcus pneumoniae proteins in resisting streptococcus pneumoniae infection, yin Yibing and the like).
The streptococcus pneumoniae with a bacterial number of 31601 is proved to be commercially available in the literature ("influence of different treatment methods on genome extraction of streptococcus pneumoniae", yellow ocean, etc.).
Streptococcus pneumoniae having accession numbers 31687 and 31759 are identified in the literature ("Streptococcus pneumoniae candidate protein vaccine ClpP conservation and antigenicity", cao Ju, etc.) as being commercially available from the China center for medical collection of bacteria (CMCC).
The streptococcus pneumoniae with the bacterial number of 31692 is proved to be purchased from the China medicine collection of bacteria (CMCC) in the literature ("study of the large-scale fermentation process conditions of streptococcus pneumoniae by an empirical amplification method", zhang Xinzhuang, etc., microbiological immunology progress, volume 44, 6, page 49 left column 1.1, section 1.3 of 2016, 12 th month).
EXAMPLE 1 preparation and evaluation of the Performance of the lyoprotectant of the invention
1. Preparation method
The freeze-drying protective agent comprises the following components: 2% soytone, 2% trehalose, 6% mannitol, 0.6% glycerol, the balance being water (except glycerol% in volume/volume ratio, the rest% in weight/volume ratio). The preparation method comprises the following steps:
(1) Raw and auxiliary materials
A Soy peptone
The calculation formula is as follows: the required amount (g) =the formulated amount (ml) ×0.02g/ml
Trehalose B
The calculation formula is as follows: the required amount (g) =the formulated amount (ml) ×0.02g/ml
C mannitol
The calculation formula is as follows: the required amount (g) =the formulated amount (ml) ×0.06g/ml
D glycerol
The calculation formula is as follows: the required amount (ml) =the formulated amount (ml) ×0.006ml/ml
(2) Weighing and dissolving reagent (100 ml for example)
A, placing weighing paper on an electronic balance, and clearing. The trehalose 2g, soytone 2g, mannitol 6g were weighed into a beaker with a stainless steel spoon, respectively.
B0.6 ml of glycerol was slowly aspirated with a pipette and slowly added to the beaker.
And C, sucking 99.4ml of water for injection into the beaker by using an electric pipette, adding a magnetic stirring rotor, placing the beaker on a magnetic stirrer, and stirring at room temperature until the water is completely dissolved.
D, preservation and expiration date: preserving at 2-8 deg.c for 24 hr, sterilizing and filtering in 24 hr, and sterilizing for one month.
2. Evaluation of Performance
2.1 Freeze-drying survival
Culturing Streptococcus pneumoniae until logarithmic phase, centrifuging a certain amount of bacterial liquid to obtain bacterial precipitate, mixing with lyophilized protectant, making into bacterial suspension, and packaging into bacterial tube with bacterial liquid of 1×10 per bottle (0.3 ml) containing viable bacteria 8 ～1×10 10 CFU/0.3ml was placed in a lyophilizer for lyophilization. The lyophilization procedure was:
(1) Pre-freezing: -50 ℃,180min; deep freezing for 60min;
(2) And (3) main drying: -40 ℃,600min; -10 ℃,300min (vacuum degree: 220 mTorr)
(3) And (5) final drying: 36 ℃,360min (vacuum degree: 100 mTorr)
Taking 1 strain tube of each serotype, adding culture medium for re-dissolving, performing 10-time gradient dilution, taking 100 μl of bacterial liquid with proper dilution, dripping into blood agar plates, uniformly coating, placing into an incubator at 36 ℃ for culturing for 18-24 h, counting after bacterial colonies grow out, calculating bacterial concentration (sum of viable bacteria Count (CFU) =plate colony count/plate count×10×dilution), and calculating survival rate (viable bacteria count after freeze-drying/viable bacteria count before freeze-drying).
10% skim milk (preparation method comprises weighing 10g of skim milk powder into a container, adding 100ml of water for injection, heating (not more than 60deg.C), stirring until skim milk powder is completely dissolved, sterilizing at 110deg.C for 15min, and lyophilizing to obtain survival rate.
Taking serotype 7F as an example, the results are shown in figure 1. Therefore, the lyoprotectant (self-made formula) provided by the invention is integrally equivalent to the lyoprotection capability of skim milk and a commercially available product.
2.2 acceleration stability
The freeze-dried strain tube in 2.1 was further taken and left at 25.+ -. 2 ℃ for 8 weeks, and the viable count was measured for weeks 0, 1, 2, 3, 4, 5, 6, 7, 8 according to the method described in 2.1. By way of example, serotype 18C was used and the results are shown in FIG. 2. The freeze-drying protective agent can still play a certain role in protecting the streptococcus pneumoniae after freeze-drying at 25 ℃.
In addition, skim milk was taken as a positive control group, and acceleration stability of the skim milk protectant at 25+ -2deg.C was examined by taking serotype 18C as an example, and as a result, the protectant in example 1 was comparable to the whole protective ability of skim milk as shown in FIG. 2.
FIG. 3 shows the change in viable count of 13 serotypes of seed lot prepared in a GMP plant when placed at 25.+ -. 2 ℃ for 32 weeks. As can be seen, the strain has not been inactivated by standing at 25.+ -. 2 ℃ for 32 weeks.
2.3 real time stability
And (3) taking the freeze-dried strain tube in the step 2.1, standing for 1 year at the temperature of 2-8 ℃, and detecting the number of viable bacteria according to the method described in the step 2.1.
The results are shown in FIG. 4. The freeze-drying protective agent has no obvious difference on the number of living bacteria after being placed for 1 year at the temperature of 2-8 ℃, which shows that the freeze-drying protective agent has good effect on protecting the activity of the streptococcus pneumoniae after freeze drying. (the figure shows only the results of the experiment for 1 year for 13 serotypes, stability experiments are underway.)
2.4 appearance evaluation test results
Good appearance and no collapse. As in fig. 5.
The results show that the freeze-drying protective agent can effectively protect the streptococcus pneumoniae activity in the freeze-drying process, and is obviously beneficial to the maintenance of the streptococcus pneumoniae activity after freeze-drying.
In addition, the freeze-drying protective agent has low cost of raw materials, namely 1555.02 yuan/kg of soytone, 3400 yuan/kg of trehalose, 205 yuan/kg of mannitol, 0.06 yuan/ml of glycerol and water for injection (neglected); the cost of the folded freeze-drying protective agent is 0.11 yuan/ml, and the selling price of the commercial product is 1.85 yuan/ml, which is far higher than that of the invention.
Example 2 lyoprotectant of the present invention and lyoprotection thereof
The freeze-drying protective agent comprises the following components: 4% soytone, 4% trehalose, 7% mannitol, 1.2% glycerol, the balance being water (the percentages being weight/volume ratios except glycerol). The preparation method is the same as in example 1.
The effect of lyoprotectants of the present invention on post-lyophilization survival of Streptococcus pneumoniae type 4 was tested as described in section 2.1 of example 1.
As a result, it was found that the survival rate of Streptococcus pneumoniae type 4 after lyophilization was 76.73% under the protection of the lyoprotectant of the present example.
Example 3 lyoprotectant of the present invention and lyoprotection thereof
The freeze-drying protective agent comprises the following components: 4% soytone, 1% trehalose, 6% mannitol, 1.2% glycerol, the balance being water (the percentages being weight/volume ratios except glycerol). The preparation method is the same as in example 1.
The effect of lyoprotectants of the present invention on post-lyophilization survival of Streptococcus pneumoniae type 4 was tested as described in section 2.1 of example 1.
As a result, it was found that the survival rate of Streptococcus pneumoniae type 4 after lyophilization was 74.01% under the protection of the lyoprotectant of the present example.
Example 4 lyoprotectant of the present invention and lyoprotection thereof
The freeze-drying protective agent comprises the following components: 1% soytone, 4% trehalose, 6% mannitol, 1.2% glycerol, the balance being water (the percentages being weight/volume ratios except glycerol). The preparation method is the same as in example 1.
The effect of lyoprotectants of the present invention on post-lyophilization survival of Streptococcus pneumoniae type 4 was tested as described in section 2.1 of example 1.
As a result, it was found that the survival rate after freeze-drying of Streptococcus pneumoniae type 4 was 78.37% under the protection of the lyoprotectant of the present example.
The beneficial effects of the present invention are further illustrated below with comparative examples.
Comparative example 1 protection of Streptococcus pneumoniae by Single-component lyoprotectants
10% skimmed milk, 8% trehalose, 8% sucrose, 8% lactose, 5% soytone, 6% mannitol, 4% glycine, 2% glycerol, 0.9% NaCl, 2% polyethylene glycol were used as lyoprotectants (water as medium), and the survival rate after lyophilization of Streptococcus pneumoniae was examined by the method of section 2.1 of example 1. The strain is serotype 7F.
After lyophilization, the survival rate under milk protection was highest, about 70%. Trehalose, mannitol and glycerol are all components of the freeze-drying protective agent, but when the freeze-drying protective agent is used alone, the survival rate of bacteria using only trehalose and soybean peptone is 10% -20%, and the survival rate of bacteria using mannitol or glycerol is 0 (figure 6).
Comparative example 2 protection of Streptococcus pneumoniae by combination of trehalose, glycerol and mannitol
Trehalose, glycerol and mannitol were formulated into a freeze-dried protectant in the proportions described in Table 2 using water as medium, and the post-freeze-drying survival rate of Streptococcus pneumoniae was tested using the method of section 2.1 of example 1. Serotype 9V is exemplified.
TABLE 2 proportions of trehalose, glycerol and mannitol
(except for the% glycerol, the% is the volume/volume ratio, and the rest% is the weight/volume ratio)
| Numbering device | Trehalose (%) | Glycerol (%) | Mannitol (%) |
| 1 | 4 | 0.5 | 4 |
| 2 | 4 | 1.25 | 6 |
| 3 | 4 | 2 | 8 |
| 4 | 6 | 0.5 | 6 |
| 5 | 6 | 1.25 | 8 |
| 6 | 6 | 2 | 4 |
| 7 | 8 | 0.5 | 8 |
| 8 | 8 | 1.25 | 4 |
| 9 | 8 | 2 | 6 |
The results are shown in fig. 7, where the survival rate of the lyophilized milk group is about 60% and the highest survival rate of the trehalose, glycerol and mannitol combination group is about 30%, which is higher than the sum of the survival rates of trehalose, glycerol and mannitol alone (less than 20%). The group numbers 1, 4, 6, 8, 9 in table 2 are not shown in fig. 7, and the corresponding survival rates are not examined because the lyophilizate profile collapses, i.e. the formulation composition cannot support the formation of the lyophilizate.
The result shows that there is a certain synergistic effect among trehalose, glycerol and mannitol, and the three can improve the freeze-drying protection effect together, but the freeze-drying protection agent has a larger difference from the freeze-drying protection agent. Therefore, in the protective agent, various components are matched with each other, so that the survival rate of streptococcus pneumoniae in the freeze-drying process can be greatly improved.
Comparative example 3 Performance of formulation consisting of trehalose, mannitol, glycerol, sodium glutamate
The formula of the freeze-drying protective agent of the comparative example is as follows: 4% trehalose, 6% mannitol, 1.2% glycerol, 0.3% sodium glutamate (except glycerol, in volume/volume ratio, the rest are weight/volume ratio), and the balance water.
The lyophilized protectant of the present comparative example, skimmed milk and lyophilized protectant of example 1 were used to freeze-dry Streptococcus pneumoniae serotypes 4 and 18C, respectively, and the viability was examined (method is the same as that of section 2.1 of example 1) and the viable count was examined for 8 weeks at 25.+ -. 2 ℃.
The survival rate results are shown in FIG. 8, and are comparable to skimmed milk under the protection of comparative examples, taking Streptococcus pneumoniae type 4 as an example.
Taking streptococcus pneumoniae 18C as an example, after the freeze-dried powder is stored for 8 weeks at 25+/-2 ℃, the streptococcus pneumoniae 18C type activity under the protection of the freeze-drying protective agent is higher and is equivalent to that of skim milk; while the lyoprotectant of this comparative example involved in lyophilized Streptococcus pneumoniae serotype 18C, which was difficult to survive stably at 25+ -2deg.C for more than 2 weeks, was significantly worse than skim milk and the lyoprotectant of the present invention, and the results are shown in FIG. 9.
The results demonstrate that lyoprotectants need to exert protective effects not only during lyophilization, but also after lyophilization. The use of the particular component of the lyoprotectant of the present invention maintains the activity of Streptococcus pneumoniae for a longer period of time than the component of comparative example 3.
Comparative example 4 Performance comparison of trehalose, mannitol, glycerol and Soytone at different ratios
Different lyoprotectants were prepared according to table 3, glycerol being measured in volume ratio and the remaining ingredients being measured in mass/volume ratio, for example serotype 4.
Table 3 protectant formulations
| Numbering device | Formulation of |
| 0# | 10% skimmed |
| 1# | 4% trehalose +6% mannitol +1.2% glycerol +1 |
| 2# | 4% trehalose +7% mannitol +1.2% glycerol +4 |
| 3# | 1% trehalose +6% mannitol +1.2% glycerol +4 |
| 4# | 1.5% trehalose +7% mannitol +0.7% glycerol +1.5 |
| 5# | 2.5% trehalose +7%Mannitol+0.7% glycerol+2.5% soytone |
After centrifugation of Streptococcus pneumoniae to obtain bacterial pellet, the protective agents described in Table 3 were added. Streptococcus pneumoniae was lyophilized using the procedure of section 2.1 of example 1.
The survival results after lyophilization are shown in table 4.
Table 4 freeze-drying survival rate
The freeze-drying survival rate results show that the combination of 1 to 4 percent of trehalose, 6 to 7 percent of mannitol, 0.6 to 1.2 percent of glycerol and 1 to 4 percent of soytone has better freeze-drying protection effect on streptococcus pneumoniae, and the protection rate of the freeze-drying survival rate is equivalent to or even higher than that of skim milk.
The results demonstrate that the lyoprotectant of the present invention has excellent lyoprotection effect.
In conclusion, the freeze-drying protective agent does not contain animal-derived components, has high survival rate after streptococcus pneumoniae is freeze-dried under the protection of the freeze-drying protective agent, has good appearance and low cost, does not obviously reduce the viable count after being placed for 1 year at 2-8 ℃, and stably survives for a long time at 25+/-2 ℃.
Claims (10)
1. A streptococcus pneumoniae animal-derived lyoprotectant is characterized in that: consists of the following components:
1 to 5 percent of soybean peptone, 1 to 5 percent of trehalose, 4 to 8 percent of mannitol, 0.3 to 1.5 percent of glycerol and the balance of water.
2. The lyoprotectant of claim 1, wherein: consists of the following components:
1 to 4 percent of soybean peptone, 1 to 4 percent of trehalose, 6 to 7 percent of mannitol and 0.6 to 1.2 percent of glycerol, and the balance of water.
3. The lyoprotectant of claim 2, wherein: consists of the following components:
1.5 to 2.5 percent of soybean peptone, 1.5 to 2.5 percent of trehalose, 6 to 7 percent of mannitol and 0.6 to 0.7 percent of glycerol, and the balance of water.
4. The lyoprotectant of claim 2, wherein: consists of the following components:
2% w/v soytone, 2% w/v trehalose, 6% w/v mannitol and 0.6% v/v glycerol, the balance being water.
5. A method for freeze-drying streptococcus pneumoniae, which is characterized by comprising the following steps: the method is characterized in that the freeze-drying protective agent in any one of claims 1-4 is mixed with bacterial precipitation of streptococcus pneumoniae to prepare bacterial suspension, and freeze-drying is carried out.
6. The method of claim 5, wherein: the serotype of streptococcus pneumoniae is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
7. A streptococcus pneumoniae lyophilized powder, characterized in that: the freeze-dried powder is a product obtained by mixing the freeze-drying protective agent according to any of claims 1-4 with bacterial precipitation of streptococcus pneumoniae to prepare bacterial suspension and freeze-drying.
8. The lyophilized powder of claim 7, wherein: the serotype of streptococcus pneumoniae is serotype 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F.
9. A streptococcus pneumoniae vaccine prepared by using the lyophilized powder of claim 7 or 8, performing fermentation purification and necessary chemical treatment, and adding pharmaceutically acceptable auxiliary materials or auxiliary components.
10. Use of a lyoprotectant according to any one of claims 1 to 4 or a lyophilized powder according to any one of claims 7 to 8 for the preparation of a streptococcus pneumoniae-related vaccine.
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| EP3668541A4 (en) * | 2017-08-16 | 2021-05-26 | Merck Sharp & Dohme Corp. | Pneumococcal conjugate vaccine formulations |
| WO2019172112A1 (en) * | 2018-03-06 | 2019-09-12 | ゼノアックリソース株式会社 | Cell cryopreservation solution and use thereof |
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