A pneumococcal conjugate combination vaccine and its preparation method

Abstract

The present invention discloses the preparation of a pneumococcal conjugate combination vaccine, belonging to the technical field of biopreparation. The composition includes 26 different pneumococcal polysaccharide-protein conjugates. The pneumococcal polysaccharides include 26 serotypes, namely 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B. The carrier protein used in the conjugate is tetanus toxoid (TT). The polysaccharide-protein conjugate stock solution is prepared by covalently binding the polysaccharide and TT through a cyanide activation method. The polysaccharide-protein conjugate stock solution is then adsorbed with aluminum adjuvant in an appropriate proportion to obtain the pneumococcal conjugate vaccine. In particular, the pneumococcal polysaccharides used in the present invention do not need to undergo a degradation reaction, effectively protecting the antigenicity of the polysaccharide. The production of three consecutive batches of conjugate stock solution and vaccine is carried out, and the conjugate yield and various verification indexes are stable. The results of non-clinical safety evaluation studies show that the vaccine has good safety and immunogenicity. The pneumococcal conjugate vaccine prepared by the present invention is used for the immunoprevention of pneumococcal infections of related serotypes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopreparation, and specifically relates to a pneumococcal polysaccharide-protein conjugate combined vaccine and a preparation method thereof. The 26-valent pneumococcal conjugate vaccine prepared by the present invention is used for the immunoprevention, diagnosis and treatment of pneumococcal infections. Background Art

[0002] Pneumococcal diseases are one of the serious global public health problems. The pathogen, Streptococcus pneumoniae, colonizes in the nasopharynx and usually does not cause clinical symptoms. When the colonization environment changes, such as a decrease in the body's resistance, respiratory virus infections such as measles or influenza, malnutrition, or being old and frail, Streptococcus pneumoniae will penetrate the mucosal defense system and cause invasive infections. Approximately 75% of invasive pneumococcal disease cases (IPD) and 83% of pneumococcal meningitis occur in children under 2 years old (WHO Position Paper 2019 Edition). Children under 2 years old are the population most susceptible to pneumococcal diseases, and their fatality rate is particularly high.

[0003] Streptococcus pneumoniae secretes a capsule that surrounds the cell wall. The capsule is the main virulence factor of Streptococcus pneumoniae. Streptococcus pneumoniae is divided into nearly 100 serotypes according to the different capsules, and the distribution of dominant serotypes varies in different periods and regions.

[0004] Pneumococcal vaccines are the most effective means of preventing pneumococcal infections. Currently, the vaccines available on the market are pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). In the 1980s, the 23-valent pneumococcal polysaccharide vaccine (PPV23) produced by Wyeth (acquired by Pfizer in 2009) had an immunization coverage rate of about 90% for the dominant serotypes in each continent around the world. However, pneumococcal capsular polysaccharide is a thymus-independent antigen (TI) antigen and cannot induce an effective immune response in children under 2 years old, and this age group is a high-risk population for pneumococcal infections. The pneumococcal polysaccharide-protein conjugate vaccine can be administered starting at 2 months of age for infants, with a total of 3 to 4 doses, which can promote a stronger immune response and memory response, and is a favorable immunization method for preventing pneumococcal infections faster and earlier. Research has shown that conjugate vaccines can be used in people of any age group, and the vaccination effect is also longer-lasting than that of polysaccharide vaccines. Therefore, the replacement of polysaccharide vaccines by conjugate vaccines has become the development trend of pneumococcal vaccines. The world's first PCV to be marketed was PCV7 (Prevnar 7) produced by Wyeth, which was approved by the FDA in 2000. In 2010, PCV13 (Prevnar 13), which can cover more serotypes, was launched and replaced Prevnar 7 for routine childhood vaccination. Currently, there are 3 PCVs that have passed WHO prequalification, namely PCV10 (Synflorix) from GSK, PCV13 (Prevenar 13) from Pfizer, and PCV10 (PNEUMOSIL) from the Serum Institute of India. In 2021, the FDA successively approved the launch of higher-valent PCVs, including Prevnar 20 (PCV20) from Pfizer and VAXNEUVANCE (PCV15) from Merck. In China, PCV13s from Watson Bio and Minhai Bio are available on the market.

[0005] Currently, the marketed pneumococcal polysaccharide-protein conjugate vaccines covalently bind polysaccharide antigens to carrier proteins through chemical methods. There are mainly two methods: the reductive amination method and the cyanogen activation method. (1) Reductive amination method. The polysaccharide is oxidized by sodium periodate, and the formed aldehyde or ketone groups react with the active amino groups of the carrier protein to form a C=N double bond, which is then reduced to a C-N single bond by a reducing agent to form a stable covalent connection. (2) Cyanogen activation method. Cyanogen groups are introduced onto the polysaccharide through cyanogen bromide (CNBr) or 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), and then covalent bonds are formed with the protein primary-NH2. With the development of conjugation techniques, based on these two conjugation methods, some new conjugation means have been developed. For example, adipic dihydrazide (ADH), a molecular arm, is introduced between the polysaccharide and the protein to promote the conjugation of the polysaccharide and the protein, or new linking groups are introduced to promote the occurrence of the conjugation reaction.

[0006] However, the literature reports that before the polysaccharide-protein conjugation reaction, the molecular size of the polysaccharide is degraded to a certain extent. Common degradation methods include physical degradation, chemical degradation, and enzymatic degradation methods. Physical degradation generally includes microwave method, radiation method, ultrasonic degradation method, and high-pressure homogenizer shearing method. Chemical degradation includes acid hydrolysis, base hydrolysis, and hydrogen peroxide degradation. Enzymatic degradation methods use specific or non-specific enzymes to degrade polysaccharides.

[0007] CN108079286A "A 13-valent pneumococcal polysaccharide-protein conjugate composition, its preparation method and application" discloses that for pneumococcal capsular polysaccharides with a K D value < 0.20 detected on a Sepharose CL-4B column, before activation and derivatization, the polysaccharide molecular size is reduced to a K D value between 0.20 and 0.50 detected on a Sepharose CL-4B column by ultrasonic degradation or high-temperature hydrolysis process at 70 - 85 °C.

[0008] CN109862908B "Multivalent pneumococcal polysaccharide-protein conjugate composition" discloses a method of hydrolysis with sodium hydroxide, hydrochloric acid, and glacial acetic acid under high-temperature conditions.

[0009] CN107810010A "Multivalent pneumococcal conjugate vaccine" discloses using a high-pressure homogenizer to reduce the molecular size of the polysaccharide.

[0010] CN103830723A "A preparation method of a pneumococcal capsular polysaccharide-protein conjugate vaccine" discloses that the polysaccharide is treated with ultrasonic waves in an ice-water bath with a power of 80 - 100 W and a frequency of 20 - 40 kHz.

[0011] The antigenicity of polysaccharides is an important indicator of immunoreactivity and immunogenicity. The larger the relative molecular mass of polysaccharides, the stronger the antigenicity. A decrease in relative molecular mass may be accompanied by a loss of antigenicity. It has been proven that each polysaccharide degradation method will destroy the specific groups and antigenic epitopes of polysaccharides while reducing the molecular size of polysaccharides, and also result in large batch-to-batch differences in the quality attributes of degraded polysaccharides. Summary of the Invention

[0012] The present invention overcomes the above problem of polysaccharide antigenicity loss. The present invention uses refined polysaccharides obtained through bacterial fermentation and polysaccharide purification and harvesting. In the polysaccharide-protein conjugation process, the refined polysaccharides do not undergo any degradation treatment and directly undergo polysaccharide activation and conjugation reactions. This use of naturally purified pneumococcal polysaccharides effectively protects the antigenicity of polysaccharides. The present invention conducts the production of conjugate bulk solutions and vaccines in three consecutive batches, and the polysaccharide yield and various verification indicators are stable, confirming the stability of the process of the present invention.

[0013] The purpose of the present invention is to provide a 26-valent pneumococcal conjugate combination vaccine.

[0014] The 26-valent pneumococcal conjugate combination vaccine of the present invention is based on the capsular polysaccharides of pneumococci and covers the most common serotypes that cause pneumococcal diseases. On the basis of the original 23-valent pneumococcal polysaccharide vaccine (with 23 serotypes including 1, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F), three serotypes, namely 6A, 24F, and 35B, are added. Among them, serotype 6A is the serotype in the 13-valent pneumococcal conjugate vaccine PCV13, and the two serotypes 24F and 35B are the two serotypes with a relatively significant increasing trend of invasive pneumococcal diseases caused by non-vaccine serotypes. According to literature reports, in recent years, serotype 24F has received extensive attention and is particularly common in cases of drug-resistant invasive pneumococcal diseases. Many countries such as Germany, the UK, Spain, Denmark, and Japan have reported that the number of cases of invasive pneumococcal diseases caused by serotype 24F has been increasing year by year. The number of cases of invasive pneumococcal diseases caused by serotype 35B has increased significantly in recent years. The immune coverage rate of the 23 original serotypes can reach about 90% on each continent globally. With the addition of the 3 new serotypes in the present invention, the immune protection range is expanded, and the coverage rate far exceeds 90%.

[0015] The technical solution of the present invention is as follows: A 26-valent pneumococcal conjugate combination vaccine, including 26 pneumococcal serotypes. The conjugate is a polysaccharide-protein conjugate prepared by chemically covalently conjugating the capsular polysaccharides of pneumococci of the corresponding serotypes with the carrier protein tetanus toxoid. The 26 polysaccharide-protein conjugate bulk solutions are then adsorbed with aluminum adjuvant in an appropriate ratio to obtain the pneumococcal conjugate vaccine.

[0016] Further, the 26-valent pneumococcal conjugate combination vaccine of the present invention includes 26 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F, and 35B.

[0017] Based on the 23-valent pneumococcal polysaccharide vaccine, three serotypes, 6A, 24F, and 35B, are added, and the immune coverage rate far exceeds 90%.

[0018] Among them, except for serotypes 2 and 3, pneumococcal capsular polysaccharides are activated with 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), and then form derivatives with adipic dihydrazide (ADH) as a spacer, and then react with the carrier protein TT under the action of carbodiimide (EDAC) to generate polysaccharide-protein conjugates;

[0019] Among them, serotypes 2 and 3 polysaccharides are directly combined with TT to generate polysaccharide-protein conjugates after being activated with CDAP.

[0020] Among them, the vaccine also contains an adjuvant, and the adjuvant is an aluminum adjuvant, and the aluminum adjuvant is aluminum phosphate adjuvant.

[0021] Another object of the present invention is to provide a preparation method of the 26-valent pneumococcal conjugate combination vaccine.

[0022] For the 26-valent pneumococcal conjugate combination vaccine of the present invention, pneumococcal polysaccharides and TT are covalently combined by the cyano activation method to prepare polysaccharide-protein conjugates. Among them, serotypes 2 and 3 polysaccharides adopt the direct combination method, that is, after the polysaccharides are activated with CDAP, they are directly combined with TT to generate polysaccharide-protein conjugates. Other serotype pneumococcal polysaccharides adopt the method of first derivatizing and then combining, that is, after the polysaccharides are activated with CDAP, they form derivatives with adipic dihydrazide (ADH) as a spacer, and then react with the carrier protein TT under the action of carbodiimide (EDAC) to generate polysaccharide-protein conjugates. The preparation method is as follows:

[0023] Step 1: After the pneumococcal polysaccharides are fermented by strains and the bacteria are removed, the polysaccharide purification process adopts CTAB precipitation, sodium chloride dissociation, hydroxyapatite chromatography, and finally freeze-drying to obtain refined pneumococcal polysaccharides;

[0024] Step 2: Serotypes 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F, and 35B pneumococcal polysaccharides are activated with CDAP, and then undergo a derivatization reaction with ADH as a spacer. After ultrafiltration purification, polysaccharide derivatives are formed, and then undergo a condensation reaction with the carrier protein TT under the action of EDAC. After ultrafiltration, polysaccharide-protein conjugates are obtained;

[0025] Among them, for CDAP: the mass ratio of polysaccharide is 0.1 - 1.0. Preferably, for 8, 9N, 9V, 10A, 20, 33F and 35B, it is 0.1 - 0.5, and for other types, it is 0.5 - 1.0;

[0026] Among them, the final concentration of ADH is 0.2 mol / L;

[0027] Among them, the pH during the reaction process is 8.0 - 9.0;

[0028] Among them, the reaction time is not less than 2 hours.

[0029] Step 3: After the type 2 and type 3 polysaccharides are activated with CDAP, they are directly subjected to a conjugation reaction with TT, and the polysaccharide - protein conjugate is obtained through ultrafiltration;

[0030] Among them, for CDAP: the mass ratio of polysaccharide is 0.1 - 1.0, preferably 0.25 - 0.75;

[0031] Among them, the mass ratio of polysaccharide to carrier protein TT is 1:0.5 - 1:2, preferably 1:1 - 1:2;

[0032] Among them, the pH during the reaction process is 8.0 - 9.0,

[0033] Among them, the reaction time is not less than 2 hours.

[0034] Step 4: The polysaccharide - protein conjugate is chromatographed on Sepharose 4FF, and the fractions near V0 and before K D 0.2 are the purified conjugate. After sterile filtration, it is the stock solution of pneumococcal polysaccharide - protein conjugate;

[0035] Step 5: The stock solutions of the 26 - type polysaccharide - protein conjugates are mixed, and then Tween 80, aluminum phosphate adjuvant, and sodium chloride solution are added. After filling, it is the 26 - valent pneumococcal conjugate combination vaccine.

[0036] Furthermore, for the refined pneumococcal polysaccharides, the weight - average molecular weight Mw of most types is 300 - 800 kDa, for type 2 and type 7F it is 900 - 1200 kDa, and for type 18C, 19A and 19F it is 100 - 400 kDa. Among them, except for types 6A, 24F and 35B, the quality of the refined polysaccharides of the other 23 types meets the polysaccharide quality standards in the "23 - valent pneumococcal polysaccharide vaccine" in the Pharmacopoeia of the People's Republic of China in 2020, and the quality of the refined polysaccharides of types 6A, 24F and 35B meets the approved requirements.

[0037] Furthermore, the derivation rate (content of ADH) of pneumococcal polysaccharide derivatives of types 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B is 1% - 10%. By controlling the addition amount of CDAP and other reaction parameters in the derivation reaction, the content of ADH is controlled, avoiding excessive cross-linking of the polysaccharide-protein conjugate and ensuring the smooth progress of chromatographic purification and sterile filtration.

[0038] For the 26-valent pneumococcal conjugate combination vaccine of the present invention, 26 kinds of polysaccharide-protein conjugate stock solutions are weighed separately and added to a Tween 80 solution and stirred evenly. The mixed stock solution is mixed with aluminum phosphate adjuvant and adsorbed overnight. The above semi-finished product is filled into a sterile 1 ml pre-filled syringe, and it becomes the finished product after filling.

[0039] Furthermore, the pH of the vaccine is 5.0 - 7.0, the aluminum ion content is 0.15 - 0.35 mg / ml, the sodium ion content is 7.5 - 9.5 g / L, and the Tween 80 content is 120 - 180 μg / ml.

[0040] Furthermore, in the combined vaccine of unit dose, the content of each type of pneumococcal polysaccharide is 2.2 μg ± 30%, and that of 6B is 4.4 μg ± 30%. The content of each dose of vaccine is 0.5 ml.

[0041] Preparation of the stock solutions of 2-valent and 3-valent pneumococcal polysaccharide-protein conjugates according to one of the examples

[0042] Weigh 1 g of polysaccharide, add 100 mg / ml CDAP acetonitrile solution (the mass ratio of CDAP to polysaccharide is 0.10 - 0.75) for activation, simultaneously add triethylamine aqueous solution to maintain pH 8.0 - 9.0, add carrier protein (the feeding ratio of polysaccharide to protein is 1:1 - 1:2), maintain pH 8.0 - 9.0, the reaction time is not less than 2 hours, and obtain the polysaccharide-protein conjugate through ultrafiltration or dialysis.

[0043] Preparation of the stock solutions of 2-valent and 3-valent pneumococcal polysaccharide-protein conjugates according to one of the examples

[0044] Preparation of the stock solutions of the other 24-valent pneumococcal polysaccharide-protein conjugates except for 2-valent and 3-valent

[0045] Weigh 1 g of polysaccharide, add 100 mg / ml CDAP acetonitrile solution (mass ratio of CDAP to polysaccharide is 0.10 - 0.75) for activation, then add aqueous triethylamine solution to adjust the pH to 8.0 - 9.5, maintain the pH range of 8.0 - 9.5, add an equal volume of ADH solution to a final concentration of 0.2 mol / L, maintain the pH range of 8.0 - 9.0, and the reaction time is not less than 2 hours. After ultrafiltration and purification, a polysaccharide derivative is obtained. Mix the polysaccharide derivative with carrier protein, add EDAC solution to a final concentration of 0.02 mol / L, maintain the pH around 5.6, and the reaction time is not less than 2 hours. After ultrafiltration and purification, a polysaccharide-protein conjugate is obtained.

[0046] Preparation of 26-valent pneumococcal conjugate vaccine according to one of the embodiments

[0047] Weigh the stock solutions of 26 polysaccharide-protein conjugates according to the content of each type of pneumococcal polysaccharide in each dose of the vaccine being 2.2 μg ± 30% and 6B being 4.4 μg ± 30%, and add them to the Tween 80 solution and stir evenly; mix the mixed stock solution with aluminum phosphate adjuvant and adsorb overnight; where the aluminum ion content is 0.15 - 0.35 mg / ml, the sodium ion content is 7.5 - 9.5 g / L, and the Tween 80 content is 120 - 180 μg / ml.

[0048] Dispense the above semi-finished product into sterile 1-ml pre-filled syringes. After filling, it is the finished product, and the content of each dose of the vaccine is 0.5 ml.

[0049] Another object of the present invention is to provide the use of the vaccine in the preparation of a drug for preventing or treating pneumonia.

[0050] The pre-clinical animal experiment results of the 26-valent pneumococcal conjugate combination vaccine of the present invention show good safety and immunogenicity. Compared with the commercially available 13-valent pneumococcal conjugate vaccine, all 26 serotypes can stimulate mice to produce high-level titers of antibodies.

[0051] Compared with the existing pneumococcal conjugate combination vaccines, the present invention has the following beneficial effects:

[0052] (1) The refined polysaccharide used in the polysaccharide-protein conjugation process of the 26-valent pneumococcal conjugate combination vaccine of the present invention does not undergo any degradation treatment and directly undergoes polysaccharide activation and conjugation reactions. This use of naturally purified pneumococcal polysaccharide effectively protects the antigenicity of the polysaccharide. The present invention conducts the production of three consecutive batches of conjugate stock solutions and vaccines, and the polysaccharide yield and various inspection indexes are stable, confirming the stability of the process of the present invention.

[0053] (2) By controlling the addition amount of CDAP and other reaction parameters in the derivatization reaction, the derivatization rate is controlled, avoiding excessive cross-linking of the polysaccharide-protein conjugate, and ensuring the smooth progress of chromatography purification and sterile filtration.

[0054] (3) The antigenicity of pneumococcal polysaccharides, conjugates and their intermediates in the preparation process was monitored by rate nephelometry, and antigenicity analysis was carried out, providing a reference basis for the selection of the pneumococcal polysaccharide-protein conjugation process route. It maximally avoids damaging the specific epitopes of the antigen in the vaccine bulk solution and protects the integrity of the antigen.

[0055] (4) The results of preclinical animal experiments showed that the 26-valent pneumococcal conjugate combination vaccine of the present invention has good safety and immunogenicity. Compared with the marketed 13-valent pneumococcal conjugate vaccine, the antibody titers of 13 serotypes are not inferior to those of the same serotypes in the 13-valent pneumococcal conjugate vaccine, and the other 13 serotypes can also stimulate mice to produce high-level antibody titers.

[0056] The raw materials used in the present invention can all be purchased on the market or prepared according to the methods of the existing technology.

[0057] The polysaccharide and the carrier protein TT were prepared by the company according to the existing methods, and the strains used were purchased from the China National Institute for the Control of Pharmaceutical and Biological Products, China Medical Bacterial Preservation Management Center. The main chemical reagents were purchased from Sigma Company.

[0058] Further explanations are made for the English abbreviations or technical terms appearing in the specification:

[0059] CTAB: hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide, is a cationic detergent that can be used to precipitate polysaccharides.

[0060] CDAP: 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate, 1-cyano-4-dimethylamino-pyridine tetrafluoroborate, CDAP can be used to activate polysaccharides.

[0061] ADH: Adipoyl Hydrazide, adipic dihydrazide, abbreviated as adipic dihydrazide. ADH is an excellent coupling agent that can chemically crosslink with aldehyde groups, etc.

[0062] Carrier protein TT: tetanus toxoid, this product is prepared by inoculating Clostridium tetani with strong toxigenicity into a suitable culture medium, inactivating and detoxifying the produced exotoxin with formaldehyde solution, and then filtering to remove bacteria. It is commonly used as a carrier protein in conjugate vaccines.

[0063] EDAC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, a carbodiimide, used as an activating reagent for carboxyl groups in amide synthesis.

[0064] Sepharose 4FF: Sepharose 4Fast Flow, a gel filtration chromatography packing material. It uses a porous gel packing material as the stationary phase, and gel filtration chromatography is a liquid chromatography method that separates each component in the order of molecular size.

[0065] V0: The external water volume, which refers to the volume of the liquid mobile phase between the gel particles in the chromatography column. Since macromolecules do not enter the interior of the gel particles but only exist in the mobile phase between the gel particles, their elution volume is equal to V0. Polysaccharide-protein conjugates belong to this type of macromolecule, so the elution peak near V0 is collected in gel chromatography.

[0066] Hydroxyapatite (CHT): Ceramic hydroxyapatite, a hydroxy compound composite mode chromatography medium with important application value, with a spherical appearance and ultra-large pore size, divided into two types: CHT I and CHTII. Detailed implementation mode

[0067] The present invention is further illustrated by the following examples.

[0068] The products and their preparation methods of the present invention are further described below in combination with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0069] Example 1: Preparation and test results of pneumococcal polysaccharide

[0070] Twenty-six pneumococcal serotype strains were sourced from the China National Center for Medical Bacterial Culture Collection, National Institutes for Food and Drug Control. The above strains were passaged to establish a library and a working seed lot was prepared. After the working seed lot was opened and inoculated into the fermenter for culture, the number of passages should not exceed 5 generations. Cultivation was terminated in the late logarithmic growth phase or the early stationary phase, and sodium deoxycholate was added to sterilize.

[0071] The supernatant was collected after centrifuging the sterilized culture solution. The supernatant was precipitated with 1% - 3% CDAB, ultrafiltered and purified, purified by hydroxyapatite (CHT II) chromatography to collect the flow-through peak, ultrafiltered and concentrated, and freeze-dried to obtain refined pneumococcal polysaccharide.

[0072] Among them, except for types 6A, 24F, and 35B, the quality of the refined polysaccharides of the other 23 types meets the polysaccharide quality standards in the "23-Valent Pneumococcal Polysaccharide Vaccine" in the Pharmacopoeia of the People's Republic of China in 2020, and the quality of the refined polysaccharides of types 6A, 24F, and 35B meets the approved requirements. The test results of the 23 serotype pneumococcal polysaccharides are shown in the following table.

[0073] Table 1 Test Results of 23 Serotype Pneumococcal Polysaccharides

[0074]

[0075]

[0076] Example 2: Preparation and Test Results of the Bulk Solution of 2-Valent and 3-Valent Pneumococcal Polysaccharide-Protein Conjugates

[0077] Weigh 1 g of polysaccharide, add 100 mg / ml CDAP acetonitrile solution (CDAP: polysaccharide mass ratio is 0.10 - 0.75) for activation, and at the same time add triethylamine aqueous solution to maintain pH 8.0 - 9.0. Add carrier protein (polysaccharide: protein feeding ratio is 1:1 - 1:2), maintain pH 8.0 - 9.0, and the reaction time is not less than 2 hours. The polysaccharide-protein conjugate is obtained through ultrafiltration or dialysis. The polysaccharide-protein conjugate is collected near V0 by Sepharose 4FF column chromatography, and the fraction before K D 0.2 is the purified conjugate. After filtration through a 0.22 μm sterile filter, it is the bulk solution of the conjugate.

[0078] Table 2 Test Results of the Bulk Solution of 2-Valent and 3-Valent Pneumococcal Polysaccharide-Protein Conjugates

[0079]

[0080] Example 3: Preparation and Test Results of the Bulk Solution of 24-Valent Pneumococcal Polysaccharide-Protein Conjugates Except for 2-Valent and 3-Valent

[0081] Weigh 1 g of polysaccharide, add 100 mg / ml CDAP acetonitrile solution (CDAP: polysaccharide mass ratio is 0.10 - 0.75) for activation, add triethylamine aqueous solution to adjust to pH 8.0 - 9.5, maintain the pH range of 8.0 - 9.5, add an equal volume of ADH solution to a final concentration of 0.2 mol / L, maintain the pH range of 8.0 - 9.0, and the reaction time is not less than 2 hours. After ultrafiltration purification, a polysaccharide derivative is obtained. The polysaccharide derivative is mixed with carrier protein, add EDAC solution to a final concentration of 0.02 mol / L, maintain pH around 5.6, and the reaction time is not less than 2 hours. After ultrafiltration purification, a polysaccharide-protein conjugate is obtained. The conjugate is purified by Sepharose 4FF column chromatography, near V0, K DComponents before 0.2 are the purified conjugate. After filtration through a 0.22 μm sterile filter, it is the conjugate stock solution. The results of the determination of the derivation rates of three consecutive batches of the 24-valent pneumococcal polysaccharide derivatives are shown in Table 3, and the results of the determination of the 24-valent pneumococcal polysaccharide-protein conjugate stock solution are shown in Table 4.

[0082] Table 3 Results of the determination of the derivation rates of the 24-valent pneumococcal polysaccharide derivatives

[0083]

[0084] Table 4 Results of the determination of the 24-valent pneumococcal polysaccharide-protein conjugate stock solution

[0085]

[0086]

[0087] Example 4: Preparation of the 26-valent pneumococcal conjugate vaccine

[0088] According to the content of each type of pneumococcal polysaccharide in each dose of the vaccine being 2.2 μg ± 30% and that of 6B being 4.4 μg ± 30%, 26 kinds of polysaccharide-protein conjugate stock solutions were weighed respectively and added to the Tween 80 solution and stirred evenly. The mixed stock solution was mixed with aluminum phosphate adjuvant and adsorbed overnight. Among them, the aluminum ion content was 0.15 - 0.35 mg / ml, the sodium ion content was 7.5 - 9.5 g / L, and the Tween 80 content was 120 - 180 μg / ml.

[0089] The above semi-finished product was filled into sterile 1 ml pre-filled syringes, and after filling, it was the finished product. The content of each dose of the vaccine was 0.5 ml.

[0090] Example 5: Study on the relative antigenicity of polysaccharides and degraded polysaccharides

[0091] In previous studies, we used the rate nephelometry method to conduct a large number of studies on the relative antigenicity of the 26-valent polysaccharides and degraded polysaccharides, and carried out acid hydrolysis and high-pressure homogenizer shearing tests respectively. (1) Taking type 4 as an example, the hydrolysis reaction was carried out at 40 °C and a final concentration of 0.5 mol / L glacial acetic acid. Setting the antigen of the refined polysaccharide as 100, the relative antigenicity of the degraded polysaccharide was measured, and the results are shown in Table 5. (2) Taking 10A and 33F as examples, the pressure of the high-pressure homogenizer was set at 1000 Bar and sheared 30 times. Samples were taken at 2, 4, 6, 8, 10, 20, and 30 times respectively to measure the relative antigenicity of the degraded polysaccharide, and the results are shown in Table 6.

[0092] The results of the relative antigenicity comparison test showed that both acid hydrolysis and high-pressure homogenizer shearing would reduce the antigenicity of polysaccharides to varying degrees. Therefore, the polysaccharides selected in the present invention were not degraded by any method.

[0093] Table 5 Research Results of Relative Antigens under Acid Hydrolysis Conditions of Type 4

[0094]

[0095] Table 6 Research Results of Relative Antigens under High-Pressure Homogenization Conditions of Type 8 and 33F

[0096]

[0097] Example 6: Research on Relative Antigenicity in Different Conjugation Methods of Type 2 and Type 3

[0098] The immunoreactivity retention degree (i.e., antigenicity) of samples during the preparation of pneumococcal polysaccharides and their polysaccharide conjugates was measured by rate nephelometry. The immunoreactivity retention value of the polysaccharide was set as 100, and the relative retention value of immunoreactivity of samples at the same concentration (such as derivatives and conjugate stock solutions) was calculated.

[0099] The results of rate nephelometry showed that the rate nephelometry results of the stock solutions of type 2 and type 3 pneumococcal polysaccharide conjugates prepared by the derivative conjugation method showed poor immunoreactivity retention. Subsequently, by changing the conjugation process, the stock solutions of type 2 and type 3 polysaccharide-protein conjugates were prepared by the direct conjugation method. The rate nephelometry results showed that the immunoreactivity retention of the conjugate stock solution was better. Two batches were selected for each type and each method for the comparison of the rate nephelometry results. See Table 7. Therefore, the direct conjugation method was finally adopted for type 2 and type 3.

[0100] Table 7 Research Results of Relative Antigenicity in Different Conjugation Methods of Type 2 and Type 3

[0101]

[0102] The rate nephelometry results of the stock solutions of type 2 and type 3 pneumococcal polysaccharide conjugates prepared by the derivative conjugation method showed poor immunoreactivity retention. Especially for type 3, the rate nephelometry result was almost 0, and the conjugation process had to be changed.

[0103] Example 7: Investigation of Process Stability

[0104] The present invention carried out the production of three consecutive batches of conjugate stock solutions and vaccines. The derivatization rates of polysaccharides in the three batches are shown in Table 3. The differences in the derivatization rates of polysaccharides of each serotype were not significant. The three batches of stock solutions produced continuously were subjected to a full analysis and detection, which met the requirements of the proposed quality standards. The total yields of the three batches of stock solutions were in good consistency, and the consistency between batches was good. Taking four types of 9N, 9V, 10A, and 11A as examples, the free polysaccharides and polysaccharide yields of three consecutive batches of stock solutions were listed. The three consecutive batches of vaccines all met the quality standards, which confirmed the stability of the process of the present invention.

[0105] Table 8 Results of Free Polysaccharides and Polysaccharide Yields of Stock Solutions of Four Types of 9N, 9V, 10A, and 11A

[0106]

[0107] Example 8: Comparison of Immunogenicity with the Commercially Available 13-Valent Pneumococcal Conjugate Vaccine

[0108] Using the domestic commercially available similar product "13-valent pneumococcal conjugate vaccine" as the control, an animal experiment was conducted to compare the immunogenicity effects. The immunization schedule adopted was as follows: Two groups of vaccines were each administered to NIH mice at a dose of 1 / 2 dose per mouse, once every 2 weeks for 3 consecutive times. The sera collected 14 days after each administration (i.e., the sera after the first, second, and third immunizations) were tested for serum specific antibody titers and seroconversion rates. The results are shown in Table 9.

[0109] Table 9 Comparison of Immunogenicity with the Commercially Available 13-Valent Pneumococcal Conjugate Vaccine

[0110]

[0111]

[0112] As can be seen from the above table, when comparing the 26-valent pneumococcal conjugate vaccine with the 13-valent pneumococcal conjugate vaccine, among the 13 common serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F), the geometric mean antibody titers of 3 serotypes, namely 1, 7F, and 14, in the sera after the third immunization were slightly lower than those of the commercially available 13-valent pneumococcal conjugate vaccine, but the geometric mean antibody titers were all within 3-fold range; the remaining 10 serotypes were not lower than those of the commercially available vaccine; for the 13 serotypes unique to the 26-valent vaccine (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 24F, 33F, 35B), all were able to stimulate mice to produce high-level antibody titers.

[0113] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A 26-valent pneumococcal conjugate combination vaccine, characterized in that: Pneumococcal capsular polysaccharides include 26 serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B. The carrier protein is TT. The pneumococcal capsular polysaccharides other than type 2 and type 3 are activated with 1-cyano-4-dimethylamino-pyridinetetrafluoroborate, and adipic dihydrazide (ADH) is used as a spacer to form derivatives, which are then reacted with the carrier protein TT under the action of carbodiimide (EDAC) to form polysaccharide-protein conjugates. Type 2 and type 3 polysaccharides are activated with CDAP and directly combined with TT to form polysaccharide-protein conjugates.

2. The vaccine according to claim 1, characterized in that The vaccine also contains an adjuvant, which is an aluminum adjuvant, and the aluminum adjuvant is an aluminum phosphate adjuvant.

3. The vaccine according to claim 1, characterized in that The Mw of types 2 and 7F is between 900 and 1200 kDa, and that of types 18C, 19A and 19F is between 100 and 400 kDa.

4. The vaccine according to claim 1, characterized in that The derivatization rates of type 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B pneumococcal polysaccharide derivatives are 1% to 10%.

5. The vaccine according to claim 1, characterized in that The free polysaccharide content of the type 4 polysaccharide-protein conjugate stock solution is not higher than 45%, and the free polysaccharide content of the other 25 serotype polysaccharide-protein conjugate stock solutions is not higher than 30%, and the free protein content is not higher than 5%.

6. The vaccine according to claim 1, characterized in that In a unit dose of the combination vaccine, the content of each type of pneumonia polysaccharide is 2.2μg±30%, and that of 6B is 4.4μg±30%; the pH of the vaccine is 5.0-7.0, the aluminum ion content is 0.15-0.35mg / ml, the sodium ion content is 7.5-9.5g / L, and the Tween 80 content is 120-180μg / ml.

7. The method for preparing the vaccine according to claim 1, characterized in that: The following steps are involved: Step 1: After the pneumococcal polysaccharide is fermented by the strain and the bacterial body is removed, the polysaccharide purification process adopts CTAB precipitation, sodium chloride dissociation, hydroxyapatite chromatography, and finally freeze-drying to obtain refined pneumococcal polysaccharide; Step 2: 1, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 24F, 33F and 35B pneumococcal polysaccharides are activated by CDAP, and then derivatized with ADH as a spacer, and then purified by ultrafiltration to form polysaccharide derivatives, and then condensed with carrier protein TT under the action of EDAC, and ultrafiltration to obtain polysaccharide protein conjugates; Step 3: After the type 2 and type 3 polysaccharides are activated by CDAP, they are directly combined with TT, and the polysaccharide-protein conjugate is obtained by ultrafiltration; Step 4: The polysaccharide-protein conjugate is chromatographed on Sepharose 4FF, and the fraction near V0 and before KD 0.2 is collected as the purified conjugate, and the purified conjugate is obtained after sterilization and filtration; Step 5: Mix the 26 types of polysaccharide-protein conjugate stock solutions and add Tween 80, aluminum phosphate adjuvant and sodium chloride solution. After filling, it becomes a 26-valent pneumococcal conjugate combination vaccine.

8. The method for preparing a vaccine according to claim 7, characterized in that In step 2, Among them, the CDAP: polysaccharide mass ratio is 0.1-1.0, Among them, the final concentration of ADH was 0.2 mol / L; Among them, the reaction process pH is 8.0-9.0; Wherein, the reaction time is not less than 2 hours; In step 3, Among them, the CDAP: polysaccharide mass ratio is 0.1-1.0, Among them, the mass ratio of polysaccharide to carrier protein TT is 1:0.5 to 1:

2. The reaction process has a pH of 8.0 to 9.

0. The reaction time is no less than 2 hours.

9. The method for preparing a vaccine according to claim 8, characterized in that In step 2, Among them, the CDAP:polysaccharide mass ratio of 8, 9N, 9V, 10A, 20, 33F and 35B was 0.1-0.5, and that of other types was 0.5-1.0; Among them, the final concentration of ADH was 0.2 mol / L; Among them, the reaction process pH is 8.0-9.0; Wherein, the reaction time is not less than 2 hours; In step 3, Among them, the CDAP:polysaccharide mass ratio is 0.25-0.75; Among them, the mass ratio of polysaccharide: carrier protein TT is 1:1 to 1:2; The reaction process has a pH of 8.0 to 9.

0. The reaction time is no less than 2 hours.

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