CN106822884B - Preparation method of multivalent pneumococcal capsular polysaccharide conjugate vaccine - Google Patents
Preparation method of multivalent pneumococcal capsular polysaccharide conjugate vaccine Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 51
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 28
- 108010060123 Conjugate Vaccines Proteins 0.000 title claims abstract description 20
- 229940031670 conjugate vaccine Drugs 0.000 title claims abstract description 20
- 238000001179 sorption measurement Methods 0.000 claims abstract description 76
- 239000002671 adjuvant Substances 0.000 claims abstract description 41
- 239000000243 solution Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 28
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229910052782 aluminium Inorganic materials 0.000 claims abstract description 24
- 229960005486 vaccine Drugs 0.000 claims abstract description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 239000001384 succinic acid Substances 0.000 claims abstract description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 6
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims abstract description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 6
- 229940068968 polysorbate 80 Drugs 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims description 16
- 230000008569 process Effects 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 6
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 108010078791 Carrier Proteins Proteins 0.000 claims description 4
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical group O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 claims description 4
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 claims description 2
- 229940031999 pneumococcal conjugate vaccine Drugs 0.000 abstract description 13
- 230000001900 immune effect Effects 0.000 abstract description 10
- 239000011550 stock solution Substances 0.000 abstract description 6
- 239000000969 carrier Substances 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 239000000872 buffer Substances 0.000 abstract description 2
- 229940031767 13-valent pneumococcal conjugate vaccine Drugs 0.000 description 16
- 239000000427 antigen Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 9
- 108091007433 antigens Proteins 0.000 description 9
- 238000003756 stirring Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 230000003053 immunization Effects 0.000 description 4
- 229940031348 multivalent vaccine Drugs 0.000 description 4
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- 238000005119 centrifugation Methods 0.000 description 2
- 206010013023 diphtheria Diseases 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 229950008882 polysorbate Drugs 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 101100027969 Caenorhabditis elegans old-1 gene Proteins 0.000 description 1
- 201000005702 Pertussis Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003497 anti-pneumococcal effect Effects 0.000 description 1
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- SPSXSWRZQFPVTJ-ZQQKUFEYSA-N hepatitis b vaccine Chemical compound C([C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC1N=CN=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)OC(=O)CNC(=O)CNC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@@H](N)CCCNC(N)=N)C1=CC=CC=C1 SPSXSWRZQFPVTJ-ZQQKUFEYSA-N 0.000 description 1
- 229940124736 hepatitis-B vaccine Drugs 0.000 description 1
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- 230000002035 prolonged effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- Medicinal Chemistry (AREA)
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- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
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Abstract
The invention relates to vaccine preparation, and particularly discloses a preparation method of a multivalent pneumococcal capsular polysaccharide conjugate vaccine, which comprises the following steps: based on the final volume of the preparation, firstly adding sodium chloride and polysorbate 80, then adding an aluminum adjuvant, preferentially adsorbing serotypes with low adsorption rate according to the adsorption rates of capsular polysaccharide-protein conjugate stock solutions of different serotypes of pneumococcus pneumoniae and the aluminum adjuvant, then adsorbing other serotypes of conjugates, and finally adding succinic acid to improve the buffer capacity of the solution. The multivalent pneumococcal capsular polysaccharide conjugate vaccine prepared by the method provided by the invention has high adsorption rate and good stability, and can exert the immune effect of the multivalent pneumococcal conjugate vaccine to the maximum extent under the condition of controlling the content of polysaccharide and protein carriers.
Description
Technical Field
The invention relates to vaccine preparation, in particular to a preparation method of a multivalent pneumococcal capsular polysaccharide conjugate vaccine.
Background
The adsorption rate of the antigen and the adjuvant is a key factor for determining the immune effect of the vaccine, and in the adsorption process, the antigen and the adjuvant are combined with each other through physical acting forces such as electrostatic force and the like, and the immune effect of the antigen can be enhanced by increasing the surface area of the antigen. Meanwhile, the vaccine adsorbed by the adjuvant can generate a 'storage effect' at an inoculation point, so that the antigen is stored in local tissues, the antigen action time is fully prolonged through the slow release of the antigen, and the immune response is improved. It is generally considered that the higher the adsorption rate of the adjuvant, the better the immunological effect, whereas the lower the adsorption rate, the worse the immunological effect. WHO requires that the adsorption rate of diphtheria and tetanus toxoid vaccines is not less than 80%, but that of pneumococcal conjugate vaccines is generally low. Therefore, based on the prior art, it is necessary to further improve the adsorption rate of the pneumococcal conjugate vaccine to improve the immune effect of the pneumococcal conjugate vaccine.
The aluminum adjuvant is one of the most widely used adjuvants at present, is the only adjuvant which is approved at present in China and can be used for human bodies, is widely used for vaccine products, and is the current hepatitis B vaccine, acellular diphtheria-pertussis-tetanus vaccine and the like in China all are adsorption vaccines containing the aluminum adjuvant.
At present, the adsorption process of the pneumococcal conjugate vaccine is rarely reported, the currently disclosed adsorption process of the aluminium adjuvant of the pneumococcal conjugate vaccine is to mix 13 pneumococcal capsular polysaccharide-protein conjugates and directly add the conjugates into the aluminium adjuvant for adsorption, and the method is easy to cause lower adsorption rate of a few serotypes and influence the immune effect.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a novel method for preparing a multivalent pneumococcal capsular polysaccharide conjugate vaccine.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
according to the first aspect, the invention provides a preparation method of a multivalent vaccine, which is sequentially adsorbed with an adjuvant step by step from low to high according to the adsorption rate of an antigen.
Specifically, the invention provides a preparation method of a multivalent pneumococcal capsular polysaccharide conjugate vaccine, and correspondingly, in the process of adsorbing a pneumococcal capsular polysaccharide-protein conjugate and an adjuvant: adsorbing the serotype pneumococcal capsular polysaccharide-protein conjugate with low adsorption rate with an aluminum adjuvant, and adsorbing other serotype pneumococcal capsular polysaccharide-protein conjugates with the aluminum adjuvant.
It should be noted that the "lower adsorption rate" is a relative concept, that is, according to the adsorption rate of each monovalent serotype pneumococcal capsular polysaccharide-protein conjugate stock solution and the aluminum adjuvant in the multivalent vaccine to be prepared, the monovalent serotype pneumococcal capsular polysaccharide-protein conjugate stock solution with the relatively lower adsorption rate is selected to be preferentially adsorbed with the aluminum adjuvant.
The preparation method mainly comprises the following steps: based on the final volume of the preparation, firstly adding sodium chloride and polysorbate 80, then adding an aluminum adjuvant, preferentially adsorbing serotypes with low adsorption rate according to the adsorption rates of capsular polysaccharide-protein conjugate stock solutions of different serotypes of pneumococcus pneumoniae and the aluminum adjuvant, then adsorbing other serotypes of conjugates, and finally adding succinic acid to improve the buffer capacity of the solution.
More specifically, the preparation method comprises the following steps in sequence:
s1, adding 0.15mol/L sodium chloride solution with the required volume into a preparation container;
s2, adding 0.5-2% of polysorbate 80 into the sodium chloride solution to obtain a solution 1, wherein the final concentration of the polysorbate 80 is 0.005-0.02%;
s3, adding an aluminum adjuvant solution with the concentration of 1-2 mg/mL into the solution 1 to obtain a solution 2, and fully stirring for 5-10 minutes to ensure that the final concentration of the aluminum adjuvant is 0.1-0.25 mg/mL;
s4, adding the serotype pneumococcal capsular polysaccharide-protein conjugate with a low adsorption rate into the solution 2, adsorbing for 30-120 minutes, adding other serotype pneumococcal capsular polysaccharide-protein conjugates, and continuously adsorbing for 16-24 hours to obtain a solution 3;
s5, adding a succinic acid solution with the concentration of 0.1-1M, pH of 5-7 into the solution 3, adjusting the pH of the system to be 5.4-7.4, enabling the final concentration of succinic acid to be 0.5-5 mM, and fully stirring for 20-30 minutes to obtain the multivalent pneumococcal capsular polysaccharide conjugate vaccine.
"final concentration" refers to the concentration of the component relative to the final vaccine preparation. In the above step, "%" means volume percent unless otherwise specified.
Further, since the vaccine prepared by the present invention is a multivalent vaccine, the pneumococcal capsular polysaccharide-protein conjugate needs to be multiple. Wherein the serotype pneumococcal capsular polysaccharide-protein conjugate with lower adsorption rate is as follows: after the adsorption rate of the various pneumococcal capsular polysaccharide-protein conjugates is measured, the adsorption rate of the various pneumococcal capsular polysaccharide-protein conjugates is lower than that of the serotypes of 30-50%.
It should be noted that the step-wise adsorption proposed by the present invention is not limited to the foregoing two-step adsorption, and may also be a multi-step adsorption. For example, after the adsorption rates of the capsular polysaccharide-protein conjugates of each monovalent serotype pneumococcal are measured, the conjugates with the lowest adsorption rate are firstly adsorbed, then the conjugates with the second lowest adsorption rate are adsorbed, and finally other conjugates are adsorbed. Therefore, the method of the present invention is not limited to the above-mentioned method, and any method may be used as long as the adsorption rate is gradually increased.
Further, the pneumococcal capsular polysaccharide-protein conjugate is a complex formed by covalently linking pneumococcal capsular polysaccharide and carrier protein. Wherein the serotype of the pneumococcal capsular polysaccharide is 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F or 33F, and the carrier protein is DT, TT or CRM197。
It should be noted that, although only the preparation method of the 13-valent pneumococcal capsular polysaccharide conjugate vaccine is specifically given in the specific embodiment, the preparation method claimed by the present invention is applicable to the preparation of other multivalent vaccines, such as 7-valent pneumococcal capsular polysaccharide conjugate vaccine, 24-valent pneumococcal capsular polysaccharide conjugate vaccine, and the like.
With the development of scientific technology, other novel serotype pneumococcal capsular polysaccharides can be discovered in the future. However, whether the serotype pneumococcal capsular polysaccharide is a new product or not, the serotype pneumococcal capsular polysaccharide belongs to the protection scope of the invention as long as the preparation method (step-by-step adsorption or the addition sequence of the reagents in the adsorption process) is utilized.
Optionally, the aluminum adjuvant is an aluminum phosphate adjuvant and/or an aluminum hydroxide adjuvant.
In a second aspect, the invention provides a multivalent pneumococcal capsular polysaccharide conjugate vaccine, wherein the adsorption rate of each serotype of pneumococcal polysaccharide in the vaccine is more than 75%.
Specifically, the vaccine is prepared by the preparation method. The method makes antigen and adjuvant fully adsorbed by stepwise adsorption technology, the pneumococcal capsular polysaccharide conjugate vaccine prepared by the method has high adsorption rate and good stability, and because protein carriers have certain adverse reactions, the protein content is reduced as much as possible in a limited range, the adsorption rate of the adjuvant is improved, and the immune effect of the pneumococcal conjugate vaccine is exerted to the maximum extent. Preferably, among the pneumococcal capsular polysaccharides of various serotypes of the vaccine, the concentration of the pneumococcal capsular polysaccharide type 6B is 8 +/-1.6 mu g/mL, and the concentration of the rest pneumococcal capsular polysaccharides of the serotypes is 4 +/-0.8 mu g/mL.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The invention has the beneficial effects that:
compared with the traditional method, the preparation method of the multivalent pneumococcal capsular polysaccharide conjugate vaccine provided by the invention avoids the defect of low adsorption rate of individual serotypes due to simultaneous adsorption of serotype pneumococcal capsular polysaccharide-protein conjugates, the serotypes with lower adsorption rate are preferentially adsorbed by using a step-by-step adsorption technology, and the adsorption process is more complete by optimizing the addition sequence of an excipient and a buffer solution, so that the adsorption rate of the pneumococcal conjugate vaccine is improved.
The multivalent pneumococcal capsular polysaccharide conjugate vaccine prepared by the method provided by the invention has high adsorption rate and good stability, and can exert the immune effect of the 13-valent pneumococcal conjugate vaccine to the maximum extent under the condition of controlling the content of polysaccharide and protein carriers.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 113 valency pneumococcal capsular polysaccharide conjugate vaccine
800mL of 13-valent pneumococcal capsular polysaccharide conjugate vaccine is prepared. The 13-valent pneumococcal capsular polysaccharide-protein conjugate in this example comprises serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19F, 19A, 23F.
Respectively adsorbing univalent serotype pneumococcal capsular polysaccharide-protein conjugate stock solution and an aluminum adjuvant, measuring the adsorption rate of the 13 serotype pneumococcal capsular polysaccharide-protein conjugate stock solutions and the aluminum adjuvant, and selecting 5 serotypes with lower adsorption rates as follows: 5. 6A, 6B, 18C, 19F.
According to the preparation method of the vaccine, the adsorption process comprises the following steps:
(1) preparing a mixed solution 1 and a mixed solution 2 of 13-valent pneumococcal capsular polysaccharide-protein conjugate, wherein the mixed solution 1 comprises serotype: 5. 6A, 6B, 18C, 19F, mix 2 comprises serotype: 1. 3, 4, 7F, 9V, 14, 19A and 23F, the concentration of each type of polysaccharide except 6B in the prepared mixed solution is 16 +/-3.2 mu g/mL, and the concentration of 6B is 32 +/-6.4 mu g/mL.
(2) 347.06mL of 0.15mol/L sodium chloride solution was added to the preparation vessel.
(3) 808 mL of 1% (V/V) polysorbate was added to give a final concentration of 0.01% (V/V). Stirred well for 10 minutes.
(4) 141.54mL of aluminum phosphate adjuvant solution with a concentration of 1.52mg/mL was added to give a final concentration of 0.25 mg/mL. Stirred well for 10 minutes.
(5) 100.14mL of the mixture 1 was added to the solution to be preferentially adsorbed, and after stirring for 60 minutes, 2195.26 mL of the mixture was added, and the stirring and adsorption were continued for 18 hours. The final concentration of each serotype polysaccharide at this time was: the concentrations of the polysaccharides of the types other than 6B were 4. + -. 0.8. mu.g/mL, and 6B was 8. + -. 1.6. mu.g/mL.
(6) Adding 8mL of succinic acid solution with the concentration of 0.1M and the pH value of 6.0 into the solution to ensure that the final concentration is 0.5mM, fully stirring and mixing, measuring the pH value to be 5.98, and fully stirring for 30 minutes to obtain the adsorbed 13-valent pneumococcal conjugate vaccine.
Comparative example 1 Effect of different adsorption methods on adsorption Rate of 13-valent pneumococcal conjugate vaccine
Comparative example 1 was set up, which differs from example 1 in that: 13 univalent serotype pneumococcal capsular polysaccharide-protein conjugate are mixed, and directly added with an aluminum adjuvant for adsorption after mixing.
The vaccines prepared in example 1 and comparative example 1 were each desorbed, and the content of each type of polysaccharide and the content of the supernatant polysaccharide after centrifugation were measured to calculate the adsorption rate. The adsorption rate (1-supernatant polysaccharide content/total sugar content after desorption) × 100%.
The adsorption rates of the various types of polysaccharides of the 13-valent pneumococcal conjugate vaccine prepared by the two methods are compared and shown in table 1. The result shows that the 13-valent pneumococcal conjugate vaccine prepared by the method has high overall adsorption rate and small difference of adsorption rate among serotypes compared with the traditional preparation method.
TABLE 113 price pneumococcal conjugate vaccine adsorption rate
Comparative example 2 Effect of different adsorption sequences on adsorption Rate of 13-valent pneumococcal conjugate vaccine
Comparative example 2 was set up, which differs from example 1 in the following steps:
(1) preparing a mixed solution 1 and a mixed solution 2 of 13-valent pneumococcal capsular polysaccharide-protein conjugate, wherein the mixed solution 1 comprises serotype: 5. 6A, 6B, 18C, 19F, mix 2 comprises serotype: 1. 3, 4, 7F, 9V, 14, 19A and 23F, the concentration of each type of polysaccharide except 6B in the prepared mixed solution is 16 +/-3.2 mu g/mL, and the concentration of 6B is 32 +/-6.4 mu g/mL.
(2) 347.06mL of 0.15mol/L sodium chloride solution was added to the preparation vessel.
(3) 8mL of succinic acid solution having a concentration of 0.1M and a pH of 6.0 was added to give a final concentration of 0.5mM, and the mixture was sufficiently stirred and mixed.
(4) 808 mL of 1% polysorbate was added to give a final concentration of 0.01%. Stirred well for 10 minutes.
(5) 141.54mL of aluminum phosphate adjuvant solution with a concentration of 1.52mg/mL was added to give a final concentration of 0.25 mg/mL. Stirred well for 10 minutes.
(6) The mixed solution 1 with a proper volume is added into the solution to be preferentially adsorbed, the mixed solution 2 is added after the mixed solution is fully stirred for 60 minutes, and the stirring and the adsorption are continued for 18 hours. Thus obtaining the adsorbed 13-valent pneumococcal conjugate vaccine.
The vaccines prepared in example 1 and comparative example 2 were each desorbed, and the content of each type of polysaccharide and the content of the supernatant polysaccharide after centrifugation were measured to calculate the adsorption rate. The adsorption rate (1-supernatant polysaccharide content/total sugar content after desorption) × 100%.
The adsorption rates of the various types of polysaccharides of the 13-valent pneumococcal conjugate vaccine prepared by the two methods are compared and shown in Table 2. The result shows that the 13-valent pneumococcal conjugate vaccine prepared by the method has high overall adsorption rate and small difference of adsorption rate among serotypes compared with the traditional preparation method.
TABLE 213 VAPOR pneumococcal conjugate vaccine adsorption Rate
Experimental example 113 price pneumococcal conjugate vaccine adsorption Rate stability investigation
In this experimental example, stability of the adsorption rate was examined using the 13-valent pneumococcal conjugate vaccine prepared in example 1. Namely, the stability of the adsorption rate of the 13-valent pneumococcal conjugate vaccine at the temperature of 2-8 ℃ is examined by measuring the adsorption rate of 13 serotype pneumococcal capsular polysaccharide-protein conjugates and aluminum adjuvants.
The specific implementation steps are as follows: the 13-valent pneumococcal conjugate vaccine is placed at the temperature of 2-8 ℃ for six months, the vaccine is taken out on the expiration day, and adsorption rate determination is carried out on each serotype after desorption, and the results are shown in table 3. The result shows that the 13-valent pneumococcal conjugate vaccine prepared by the method has good adsorption rate stability.
TABLE 3 stability study of adsorption Rate of polysaccharide of each serotype
| Serotype | 0 month | 3 months old | Serotype | 0 month | 3 months old |
| 1 | 83.21 | 80.05 | 9V | 82.08 | 80.39 |
| 3 | 85.51 | 82.32 | 14 | 85.20 | 85.15 |
| 4 | 86.67 | 85.14 | 18C | 78.25 | 77.63 |
| 5 | 81.36 | 80.33 | 19A | 82.60 | 80.22 |
| 6A | 78.66 | 77.46 | 19F | 80.58 | 79.58 |
| 6B | 80.32 | 79.28 | 23F | 86.84 | 85.12 |
| 7F | 86.15 | 84.56 |
Experimental example 213-valent pneumococcal conjugate vaccine immunogenicity examination
In this example, the 13-valent pneumococcal conjugate vaccine prepared in example 1 was used for immunogenicity examination. After the mice are immunized with the 13-valent pneumococcal conjugate vaccine, the serum of the mice is tested to resist IgG antibody of capsular polysaccharide of specific serotype streptococcus pneumoniae by an enzyme-linked immunosorbent assay (ELISA).
The specific implementation scheme is as follows: injection site: subcutaneous injection; immunization procedure: 0. immunizing at 14 and 28 days, collecting blood from orbit at 35 days, centrifuging, and collecting serum; immunization dose: 0.1 mL/mouse, and the results are shown in Table 4.
TABLE 413 VALENT VARIATION OF ANTIBODY VARIANTS IN pneumococcal conjugate VACCINE IMMUNIZED mice
| Serotype | Antibody titer | Serotype | Antibody titer |
| 1 | 5120 | 9V | 5120 |
| 3 | 1280 | 14 | 2560 |
| 4 | 5120 | 18C | 2560 |
| 5 | 2560 | 19A | 5120 |
| 6A | 1280 | 19F | 2560 |
| 6B | 2560 | 23F | 1280 |
| 7F | 2560 |
The result of the embodiment shows that after the 13-valent pneumococcal conjugate vaccine prepared by the method is used for immunizing a mouse, the titer of the anti-pneumococcal capsular polysaccharide specific antibody of each serotype is higher, namely the vaccine has better immune effect.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. A preparation method of a multivalent pneumococcal capsular polysaccharide conjugate vaccine is characterized in that in the process of adsorbing a pneumococcal capsular polysaccharide-protein conjugate and an adjuvant: adsorbing the serotype pneumococcal capsular polysaccharide-protein conjugate with a low adsorption rate with an aluminum adjuvant, and adsorbing other serotype pneumococcal capsular polysaccharide-protein conjugates with the aluminum adjuvant; the method specifically comprises the following steps:
s1, adding 0.15mol/L sodium chloride solution into a preparation container;
s2, adding 0.5-2% of polysorbate 80 into the sodium chloride solution to obtain a solution 1, wherein the final concentration of the polysorbate 80 is 0.005-0.02%;
s3, adding an aluminum adjuvant solution with the concentration of 1-2 mg/mL into the solution 1 to obtain a solution 2, wherein the final concentration of the aluminum adjuvant is 0.1-0.25 mg/mL;
s4, preparing a mixed solution 1 and a mixed solution 2 of 13-valent pneumococcal capsular polysaccharide-protein conjugate, wherein the mixed solution 1 comprises 5 serotypes with low adsorption rates: 5. 6A, 6B, 18C, 19F, mix 2 comprises serotype: 1. 3, 4, 7F, 9V, 14, 19A and 23F, adding the mixed solution 1 into the solution 2, adsorbing for 30-120 minutes, adding the mixed solution 2, and continuously adsorbing for 16-24 hours to obtain a solution 3;
s5, adding a succinic acid solution with the concentration of 0.1-1M, pH of 5-7 into the solution 3, adjusting the pH of the system to be 5.4-7.4, and enabling the final concentration of succinic acid to be 0.5-5 mM to obtain the polyvalent pneumococcal capsular polysaccharide conjugate vaccine;
the pneumococcal capsular polysaccharide-protein conjugate is multiple; the pneumococcal capsular polysaccharide-protein conjugate is a complex formed by covalently connecting pneumococcal capsular polysaccharide and carrier protein, wherein the serotypes of the pneumococcal capsular polysaccharide are 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, and the carrier protein is DT, TT or CRM197；
The aluminum adjuvant is an aluminum phosphate adjuvant and/or an aluminum hydroxide adjuvant.
2. A multivalent pneumococcal capsular polysaccharide conjugate vaccine, which is prepared by the preparation method of claim 1.
3. The vaccine of claim 2, wherein the vaccine comprises a plurality of serotype pneumococcal capsular polysaccharides, wherein the concentration of the type 6B pneumococcal capsular polysaccharide is 8 ± 1.6 μ g/mL, and the concentration of the remaining serotype pneumococcal capsular polysaccharide is 4 ± 0.8 μ g/mL.
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