WO2025098327A1 - Multivalent streptococcus pneumoniae capsular polysaccharide-protein conjugate composition, preparation method therefor, and use thereof - Google Patents

Abstract

Disclosed are a multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition and a preparation method therefor. The Streptococcus pneumoniae serotype is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F, and 33F. The composition can effectively stimulate the production of helper T cells in the body, promote the activation of B cells, and result in immune memory, so as to enable the antibody class to switch from IgM to IgG and enhance immunogenicity, thereby enabling the infants under 2 years of age to produce effective immune responses against corresponding diseases.

Description

A multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition and its preparation method and application Technical Field

The present invention relates to a multivalent pneumococcal capsular polysaccharide-protein conjugate composition and a preparation method and application thereof, and in particular to a 24-valent pneumococcal capsular polysaccharide-protein conjugate composition.

Background Art

Pneumococcal disease (PD) is one of the most serious public health problems in the world. Pneumococcus (Streptococcus pneumoniae, Spn) is the main pathogen that causes serious diseases such as pneumonia, meningitis, and bacteremia in children, and is also a common cause of acute otitis media and sinusitis.

The vaccines currently on the market include the following types: Pneumococcal vaccines include pneumococcal polysaccharide vaccine (PPV) and pneumococcal conjugate vaccine (PCV). The former mainly includes PPV23, and the latter mainly includes PCV7, PCV10, PCV13, PCV15 and PCV20.

Pneumococcal conjugate vaccines are currently the mainstream products on the market, including Pfizer, Glaxo, etc., among which protein carriers are used in two ways: single and mixed carriers. Taking Pfizer as an example, the single carrier PCV7 vaccine (PREVNAR) contains capsular polysaccharides from the 7 most popular serotypes: 4, 6B, 9V, 14, 18C, 19F and 23F. Different capsular polysaccharides are combined with CRM _197. PCV13 vaccine also uses a single carrier, which is CRM _197. In mixed carriers, two or more protein carriers are used, and certain specific serotypes are combined with specific protein carriers. For example, Glaxo's SYNFLORIX, serotypes 1, 4, 5, 6B, 7F, 9V, 14 and 23F are conjugated with Haemophilus influenzae (H.influenzae) protein D. serotype 18C is conjugated to tetanus toxoid, and serotype 19F is conjugated to diphtheria toxoid.

With the listing and promotion of the above-mentioned 13-valent products, the number of diseases caused by the serotypes included in the existing 13-valent vaccines has decreased overall, while other non-vaccine serotypes that were previously relatively less widespread have gradually received attention, so it is urgent to develop a new pneumonia vaccine to cope with environmental changes. In the development of new vaccines, it is necessary to add more serotypes to improve protection, and at the same time, the selection preference of different serotype capsular polysaccharides for carrier proteins must be considered, and the influence of combining multiple polysaccharide-protein conjugates on the immune effect of different serotypes of pneumococci. Based on this, the present invention conducts further research on the above situation.

Summary of the invention

The present invention provides a multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition, a preparation method and application thereof.

Specifically, the present application provides a multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition, which comprises 24 different Streptococcus pneumoniae capsular polysaccharide-carrier protein conjugates, wherein the Streptococcus pneumoniae serotype is selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F, and the carrier protein comprises a first carrier protein and a second carrier protein, the first carrier protein and the second carrier protein are different, and the serotype 12F can be conjugated to the first carrier protein or the second carrier protein.

Specifically, the pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 9N, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and 7F, 15B, 17F, 19F are conjugated to a second carrier protein; or, the pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 19F is conjugated to a second carrier protein; or, the pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 17F, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 15B, 19F is conjugated to a second carrier protein; or, the pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 17F, 19F is conjugated to a second carrier protein; or, the pneumococcal capsular polysaccharide serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 15B, 17F, 19F is conjugated to a second carrier protein. The serotype 12F can be conjugated to the first carrier protein or the second carrier protein.

Preferably, the pneumococcal capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 9N, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 12F, 15B, 17F, 19F are conjugated to a second carrier protein.

Preferably, the pneumococcal capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 12F, 19F are conjugated to a second carrier protein.

Preferably, the pneumococcal capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 17F, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 15B, 19F are conjugated to a second carrier protein.

Preferably, the pneumococcal capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 17F, 19F are conjugated to a second carrier protein.

Preferably, the pneumococcal capsular polysaccharide 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the pneumococcal capsular polysaccharide 7F, 9N, 15B, 17F, 19F are conjugated to a second carrier protein.

Specifically, the carrier protein should be non-toxic, capable of binding to the pneumococcal capsular polysaccharide and causing a significant increase in the immunogenicity of the polysaccharide, and easy to obtain in large quantities.

Specifically, the first carrier protein or the second carrier protein is selected from: CRM ₁₉₇ , TT (Tetanus toxoid), PD (Haemophilus influenzae protein D), pertussis toxoid (PT), and fHbp.

Specifically, the first carrier protein is CRM ₁₉₇ , and the second carrier protein is TT or PD or PT.

Preferably, the first carrier protein is CRM ₁₉₇ , and the second carrier protein is TT.

Specifically, the carrier protein of the present invention is obtained through the steps of bacterial fermentation, ultrafiltration, purification, detoxification, etc.

The CRM ₁₉₇ of the present invention is produced by Corynebacterium diphtheriae infected with the non-toxigenic phage β197 ^tox- (created by nitrosoguanidine mutagenesis of toxigenic β-rod-shaped phage). The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene (guanine to adenine). This single base change results in an amino acid substitution (glutamic acid for glycine) in the mature protein and eliminates the toxicity of diphtheria toxin.

The TT (Tetanus toxoid) and DT (diphtheria toxoid) described in the present invention have molecular weights of 151 kilodaltons (kD) and 58 kD, respectively, and are exotoxins produced by Bacillus tetani and Corynebacterium diphtheriae. They are detoxified with formaldehyde to make them lose their toxicity, and then purified by chemical methods to remove invalid protein impurities to obtain high-purity refined toxoids. They have been used as vaccines for the prevention of tetanus and diphtheria for many years. Due to their good immunogenicity and safety, they were first selected as carrier molecules at the beginning of the development of polysaccharide protein conjugate vaccines. Toxoids as carrier proteins also have certain limitations. For example, the requirements for the culture conditions of Bacillus tetani in vaccine production are relatively harsh, and a large amount of toxin supernatant needs to be processed during preparation. In addition to the possibility of residual toxicity and toxicity reversal, the toxins that have been detoxified also undergo certain conformational changes or modifications in the protein structure, which may destroy some T cell antigen sites, thereby affecting immunogenicity.

The PD described in the present invention is a surface lipoprotein with a relative molecular mass of 42,000, which exists in all Hi strains and is highly conservative _.

Specifically, the present invention also provides a method for preparing the multivalent pneumococcal capsular polysaccharide-protein conjugate composition, which comprises separating and purifying the capsular polysaccharides of 24 different serotypes (1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) of pneumococcal capsular polysaccharides, and then covalently linking the capsular polysaccharides of each serotype to a carrier protein to form a monovalent conjugate, and finally mixing the monovalent conjugates of each type to obtain a multivalent pneumococcal capsular polysaccharide-protein conjugate composition.

Specifically, the steps of separating and purifying the capsular polysaccharide of Streptococcus pneumoniae are as follows:

A pneumococcal strain library was established, and working seeds were used for pneumococcal fermentation; an appropriate amount of sodium deoxycholate was added to the pneumococcal fermentation broth for lysis; the fermentation broth after lysis was centrifuged or filtered to remove the bacteria, and the centrifugal supernatant or filtrate was collected, and then ultrafiltration and concentration were performed; the fermentation recovery liquid obtained by centrifugation or filtration was purified by acid precipitation or alcohol precipitation and column chromatography to remove the protein and cell debris that had not been precipitated during centrifugation. The filtrate was concentrated on a 100kDa membrane package and the concentrate was dialyzed and filtered to obtain a sample. After ultrafiltration, the ultrafiltration recovery liquid was filtered through a 0.2μm filter. The filtrate was subjected to in-process quality control (appearance, protein, nucleic acid content, endotoxin, molecular weight and total amount of polysaccharides). The purified polysaccharide was sterile filtered and stored at -20°C.

Specifically, the polysaccharide of the present invention can also be purified by other purification techniques well known in the art, such as column chromatography, etc. to purify the pneumococcal capsular polysaccharide.

Specifically, the composition of the present invention further comprises optional excipients and/or aluminum-based adjuvants.

The aluminum-based adjuvants include aluminum oxide, aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc. The aluminum-based adjuvants are commercially available and can also be prepared by conventional methods in the art. The excipients are preferably sodium chloride and/or succinic acid, etc.

The present invention also provides use of the multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition in the preparation of a drug for preventing or treating a disease caused by Streptococcus pneumoniae.

Specifically, the polyvalent pneumococcal polysaccharide-protein conjugate composition can be a spray, an injection, a lyophilized agent, a capsule, a tablet or a pill, etc. Immunization can be performed by the following methods: skin, injection (including subcutaneous injection, intramuscular injection, intravenous injection), oral administration, nasal drops or mucosal spray, etc.

Specifically, the preparation method of the multivalent pneumococcal polysaccharide-protein conjugate composition comprises: respectively adsorbing each type of monovalent conjugate with an aluminum adjuvant and then mixing; or mixing each type of monovalent conjugate and then adsorbing with an aluminum adjuvant to obtain a 24-valent pneumococcal capsular polysaccharide conjugate vaccine.

The multivalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can be used to protect or treat patients with pneumonia caused by pneumococcal infection. The vaccine can be injected intramuscularly, subcutaneously, intradermally or administered through mucosa, and is suitable for vaccination of humans and animals.

The beneficial effects of the present invention include:

In the first aspect, the present invention is aimed at the prevalence of serotypes of Streptococcus pneumoniae unique to my country, and creatively selects serotype combinations of 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, thereby developing a multivalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition designed for the prevalence of serotypes of Streptococcus pneumoniae unique to my country.

In the second aspect, the present invention further studies 24 different serotypes, conducts immunogenicity studies on different serotypes and carrier protein combinations, and simultaneously studies the immunogenicity of 24 polysaccharide-carrier protein compositions to screen out the best combination of polysaccharide combined with carrier protein.

Thirdly, the present invention further studies 24 different serotypes and rationally controls the content of carrier protein while ensuring maximum immunogenicity.

Fourthly, the composition of the present invention has multiple immunogenicity and protective properties against the invasion of the above-mentioned 24 serotypes of Streptococcus pneumoniae, which is superior to the low-priced pneumococcal composition already on the market, and the immune response is higher than that of the uncombined composition.

In the fifth aspect, the immunogenicity of the polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention is much higher than that of similar polyvalent polysaccharide vaccines. The composition of the present invention can effectively stimulate the body It produces helper T cells, promotes B cell activation and produces immune memory, enables the conversion of antibody classes from IgM to IgG, improves immunogenicity, and enables infants under 2 years old to produce effective immune responses against corresponding diseases.

In a sixth aspect, the multivalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can prevent invasive diseases caused by the above 24 serotypes of pneumococcus, which is more convenient, multi-effective and has lower cost.

In the seventh aspect, the polyvalent pneumococcal capsular polysaccharide-protein conjugate composition is completely adsorbed, has stable physical and chemical properties, and does not interfere with each other. Animal experiments have shown that the polyvalent pneumococcal capsular polysaccharide conjugate vaccine of the present invention can effectively induce the body to produce high-titer, specific antibodies against the above-mentioned 24 serotype capsular polysaccharides. In vitro experiments have shown that the antibodies induced by the polyvalent conjugate vaccine have obvious reactivity and neutralizing activity; in particular, the immune effect of each serotype capsular polysaccharide in the composition is not affected by another component.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Immunogenicity of the conjugates obtained by combining type 2 capsular polysaccharide with different carrier proteins;

Figure 2: Immunogenicity of the conjugates obtained by combining type 8 capsular polysaccharide with different carrier proteins;

Figure 3: Immunogenicity of the conjugates obtained by combining 9N type capsular polysaccharide with different carrier proteins;

Figure 4: Immunogenicity of the conjugates obtained by combining type 10A capsular polysaccharide with different carrier proteins;

Figure 5: Immunogenicity of the conjugates obtained by combining type 11A capsular polysaccharide with different carrier proteins;

Figure 6: Immunogenicity of the conjugates obtained by combining 12F type capsular polysaccharide with different carrier proteins;

Figure 7: Immunogenicity of the conjugates obtained by combining type 15B capsular polysaccharide with different carrier proteins;

Figure 8: Immunogenicity of the conjugates obtained by combining 17F type capsular polysaccharide with different carrier proteins;

Figure 9: Immunogenicity of the conjugates obtained by combining type 20 capsular polysaccharide with different carrier proteins;

Figure 10: Immunogens of the conjugates obtained by combining 22F type capsular polysaccharide with different carrier proteins sex;

Figure 11: Immunogenicity of the conjugates obtained by combining 33F type capsular polysaccharide with different carrier proteins;

Figure 12: Antibody titers of 24-valent pneumococcal capsular polysaccharide carrier protein conjugate vaccine against serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F;

Figure 13: Antibody titers of 24-valent pneumococcal capsular polysaccharide carrier protein conjugate vaccine against serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F, respectively.

DETAILED DESCRIPTION

The technical solutions in the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: Preparation of Streptococcus pneumoniae capsular polysaccharide stock solution

1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F serotypes of pneumococcus (Streptococcus pneumoniae) strains are all from the National Center for Medical Culture Collections (CMCC) of China. The above strains are subcultured to establish the original seed bank, the main seed bank and the working seed bank. After the working seed batch is opened, it is cultured in the inoculation fermenter, and each generation of seeds is freeze-dried and preserved. Take the working seed and pick the strain on the culture medium, inoculate it in 5ml of liquid comprehensive culture medium, culture it at 35℃±2℃, transfer it to 500ml of culture solution, and then culture and expand it to 10L culture solution and 50L culture solution. The culture was terminated at the end of the logarithmic growth phase or the beginning of the stationary phase, and samples were taken for a pure bacterial test. An appropriate amount of sterile sodium deoxycholate is added to the harvested culture fluid to lyse the bacterial cells and release the capsular polysaccharide. The sterilized culture fluid is centrifuged and the supernatant is collected and concentrated by ultrafiltration. The supernatant is further ultrafiltered and purified to obtain 24 purified serotypes of pneumococcal capsular polysaccharides, 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

Example 2: Preparation of 24 capsular polysaccharide-protein conjugate compositions and immunogenicity testing

The 24 pneumococcal capsular polysaccharide stock solutions prepared in Example 1 were conjugated with different proteins respectively.

Capsular polysaccharides of pneumococcal serotypes 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A and 23F were purified by acetic acid hydrolysis or hydrogen peroxide hydrolysis or high pressure degradation to reduce the molecular weight of the polysaccharide, and then hydrolyzed/degraded by sodium periodate or CDAP activation, and combined with CRM197.

The capsular polysaccharides of serotypes 7F and 19F of pneumococci were purified by acetic acid hydrolysis or hydrogen peroxide hydrolysis or high pressure degradation to reduce the molecular weight of the polysaccharides, and then the polysaccharides were hydrolyzed/degraded by CDAP activation and bound to TT.

Capsular polysaccharides of pneumococcal serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F were hydrolyzed with acetic acid or hydrogen peroxide or degraded under high pressure to reduce the molecular weight of the purified polysaccharides, and then activated and hydrolyzed/degraded by sodium periodate or CDAP and combined with CRM197, TT and PD protein carriers, respectively.

The polysaccharide-protein conjugate is analyzed by quality, and the free polysaccharide content is controlled within 30%, the free protein content is within 5%, the polysaccharide-protein ratio is between 0.3-2.5, and the endotoxin and other impurities are controlled within a safe and acceptable range.

The immunogenicity of pneumococcal capsular polysaccharides of serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F combined with CRM197, TT, and PD carrier proteins was tested, and the results are shown in Figures 1 to 11.

The results showed that for serotypes 2, 8, 10A and 11A, the immunogenicity was stronger when combined with the CRM197 carrier protein than with the TT carrier protein.

Example 3: Preparation of a polyvalent Streptococcus pneumoniae capsular polysaccharide-protein conjugate composition and detection of its immunogenicity

Based on the previous 13-valent pneumonia combination research composition, 1, 3, 4, 5, 6A, 6B, 9V, 14, 18C, 19A and 23F were selected to combine with CRM197, and 7F and 19F were combined with TT. Other serotypes were further screened for specific carrier proteins in the composition based on the experimental results of Example 2, as shown in Table 1 below (the CRM in the table specifically refers to CRM197).

Table 1. Screening of new serum type carrier proteins in polysaccharide-protein conjugate compositions

Each of the polysaccharide-protein monovalent conjugate stock solutions 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F prepared above is diluted with a buffer solution of pH 5.3 to 6.8 and then mixed, and then an aluminum phosphate adjuvant is added and stirred to prepare a semi-finished product, wherein the polysaccharide content in each conjugate is 2 to 8 micrograms per milliliter, and the aluminum phosphate adjuvant content is 0.125 to 0.5 mg.

Immunization experiment plan: The polyvalent vaccine stock solution was added to aluminum phosphate adjuvant to prepare immune antigens, and 2.0-2.5 kg New Zealand white rabbits were selected for thigh muscle immunization, 4 rabbits per group, each immunization dose was 0.25 ml, and blood was drawn on the 35th day to evaluate the immunogenicity of its polysaccharide. The immunization results are shown in Figures 12 and 13.

The results showed that different combinations of carrier proteins had different immune effects. The immune effects of groups 2-7, i.e., PCV24 polysaccharide conjugate vaccine compositions with some serotypes combined with TT carrier proteins, were better than those of group 1, where all carrier proteins were CRM197. The overall immunity of the composition was higher when serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9N, 9V, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F were conjugated with CRM197, 7F, 15B, 17F, 19F were conjugated with TT, and 12F was conjugated with CRM197 or TT.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The foregoing embodiments and methods described in the present invention may vary based on the abilities, experiences and preferences of those skilled in the art. The present invention merely lists the steps of the method in a certain order and does not constitute any limitation on the order of the steps of the method.

Claims (10)

A multivalent pneumococcal capsular polysaccharide-protein conjugate composition, comprising pneumococcal capsular polysaccharides from 24 different serotypes and a carrier protein, wherein the pneumococcal capsular polysaccharides from different serotypes are selected from 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19F, 19A, 20, 22F, 23F and 33F, and the carrier protein comprises a first carrier protein and a second carrier protein, and the first carrier protein and the second carrier protein are different; The serotype 12F polysaccharide is conjugated to the first carrier protein or the second carrier protein, the serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 9N, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F polysaccharides are conjugated to the first carrier protein, and the serotypes 7F, 15B, 17F, 19F polysaccharides are conjugated to the second carrier protein; or, the serotype 12F polysaccharide is conjugated to the first carrier protein or the second carrier protein, and the serotype 1 , 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 20, 22F, 23F and 33F polysaccharides are conjugated to the first carrier protein, and the polysaccharides of serotypes 7F, 9N, 19F are conjugated to the second carrier protein; or, the polysaccharide of serotype 12F is conjugated to the first carrier protein or the second carrier protein, and the polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 17 The polysaccharide of serotype F, 18C, 19A, 20, 22F, 23F and 33F is conjugated to the first carrier protein, and the polysaccharide of serotype 7F, 9N, 15B, 19F is conjugated to the second carrier protein; or, the polysaccharide of serotype 12F is conjugated to the first carrier protein or the second carrier protein, and the polysaccharide of serotype 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 18C, 19A, 20, 22F, 23F and 33F is conjugated to the first carrier protein. protein, the polysaccharide of serotype 7F, 9N, 17F, 19F is conjugated to a second carrier protein; or, the polysaccharide of serotype 12F is conjugated to a first carrier protein or a second carrier protein, the polysaccharide of serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F is conjugated to a first carrier protein, and the polysaccharide of serotypes 7F, 9N, 15B, 17F, 19F is conjugated to a second carrier protein. The composition according to claim 1, characterized in that the polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 9N, 10A, 11A, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the polysaccharides of serotypes 7F, 12F, 15B, 17F, 19F are conjugated to a second carrier protein; or, the polysaccharide of serotype 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 14, 15B, 17F, 18C, 19A, 20, 22F, 23F and 33F is conjugated to a first carrier protein, and the polysaccharide of serotype 7F, 9N, 12F, 19F is conjugated to a second carrier protein; or, the polysaccharide of serotype 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 17F, 18C, 19A, 20, 22F, 23F and 33F is conjugated to a first carrier protein, and the polysaccharide of serotype 7F, 9N, 15B, 19F is conjugated to a second carrier protein; or, the polysaccharide of serotype 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 15B, 18C, 19A, 20, 22F, 23F and 33F is conjugated to a first carrier protein, and the polysaccharide of serotype 7F, 9N, 17F, 19F is conjugated to a second carrier protein; Or, the polysaccharides of serotypes 1, 2, 3, 4, 5, 6A, 6B, 8, 9V, 10A, 11A, 12F, 14, 18C, 19A, 20, 22F, 23F and 33F are conjugated to a first carrier protein, and the polysaccharides of serotypes 7F, 9N, 15B, 17F, 19F are conjugated to a second carrier protein. The composition according to claim 1 or 2, characterized in that the first carrier protein and the second carrier protein are selected from: CRM197, TT, PD, PT, fHbp. The composition according to claim 3, characterized in that the first carrier protein is CRM197, and the second carrier protein is TT, PD or PT; preferably, the first carrier protein is CRM197, and the second carrier protein is TT. The composition according to any one of claims 1 to 4, characterized in that the composition further comprises an excipient and/or an aluminum-based adjuvant. The composition according to any one of claim 5, characterized in that the aluminum-based adjuvant is selected from aluminum oxide, aluminum hydroxide, aluminum phosphate and aluminum sulfate. The composition according to any one of claims 1 to 6, characterized in that the composition is prepared as a spray, an injection, a lyophilized agent, a capsule, a tablet or a pill. The composition according to any one of claims 1 to 7, characterized in that the composition can be immunized by the following methods: skin, injection, oral administration, nasal drops or mucosal spray; preferably, the injection is subcutaneous injection, intramuscular injection or intravenous injection. A method for preparing a multivalent pneumococcal capsular polysaccharide-protein conjugate composition as claimed in any one of claims 1 to 8, characterized in that the method comprises: adsorbing each type of monovalent conjugate with an aluminum adjuvant and then mixing them, or mixing each type of monovalent conjugate and then adsorbing them with an aluminum adjuvant to prepare a 24-valent pneumococcal capsular polysaccharide conjugate vaccine. Use of any one of the multivalent pneumococcal capsular polysaccharide-protein conjugate compositions of claims 1 to 8 in the preparation of a vaccine or a drug for preventing or treating a disease, characterized in that the disease is a disease caused by pneumococcal infection.