Pneumococcal polysaccharide-protein CDAP conjugates, methods of making and use thereof

Abstract

The application provides a pneumococcal polysaccharide-protein CDAP conjugate and a preparation method and application thereof, and relates to the technical field of vaccines. The preparation method of the pneumococcal polysaccharide-protein CDAP conjugate provided by the application uses DABCO as a buffer system, can accurately maintain the optimal pH interval of CDAP activation reaction, reduces side reactions, improves the yield, purity and batch consistency of the conjugate, and is low in cost, simple in process and suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a pneumococcal polysaccharide-protein CDAP conjugate, its preparation method, and its application. Background Technology

[0002] Streptococcus pneumoniae is a major pathogen causing serious diseases such as pneumonia, bacteremia, and meningitis, posing a significant threat, especially to children, the elderly, and those with weakened immune systems. Pneumococcal conjugate vaccines, by covalently binding pneumococcal capsular polysaccharides to carrier proteins, significantly enhance the immunogenicity of the polysaccharides, inducing the body to produce long-lasting protective antibodies. This is one of the most effective means of preventing Streptococcus pneumoniae infection.

[0003] The CDAP (1-cyano-4-(dimethylamino)pyridine tetrafluoroborate) method is currently the mainstream method for polysaccharide-protein conjugation. Its core principle is to utilize CDAP to activate the hydroxyl groups in the polysaccharide molecule, forming active cyanate groups, which then covalently bind to the amino groups of the carrier protein, forming a stable polysaccharide-protein conjugate. This method has advantages such as mild reaction conditions, high conjugation efficiency, and few byproducts, and has been widely used in the research and development and production of pneumococcal conjugate vaccines.

[0004] CDAP activation requires strict control over the pH of the reaction system, with the optimal pH range being 8.5-9.0. Within this range, CDAP activation efficiency is highest, and polysaccharide degradation and protein denaturation are effectively avoided. Currently, commonly used buffer systems in CDAP activation reactions include HEPES buffer and borate buffer, but these traditional buffer systems have significant drawbacks: HEPES buffer is expensive and has potential toxicity, making it unsuitable for large-scale industrial production; borate buffer readily forms complexes with hydroxyl groups in polysaccharides, interfering with the CDAP activation process, increasing the amount of byproducts, and reducing the yield and purity of the conjugate.

[0005] Triethylenediamine (DABCO, 1,4-diazabicyclo[2.2.2]octane) is a bicyclic tertiary amine compound with a pKa value of approximately 8.7, which highly matches the optimal pH range for CDAP activation, exhibiting excellent buffering capacity. Furthermore, the DABCO molecule lacks hydroxyl, primary, or secondary amino groups, thus avoiding competitive reactions with CDAP or the activated polysaccharide and effectively reducing byproduct formation. Currently, DABCO is primarily used as a catalyst, curing agent, and oxygen scavenger; there are no reports of its application as a buffer system in the pneumococcal polysaccharide-protein CDAP conjugation reaction, nor is there any related patent protection. Therefore, developing a pneumococcal polysaccharide-protein CDAP conjugate using DABCO as a buffer system and its preparation method, addressing the technical limitations of traditional buffer systems, has significant practical importance and application value.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for preparing pneumococcal polysaccharide-protein CDAP conjugates, thereby overcoming the shortcomings of existing technologies in CDAP binding reactions, such as poor pH stability, numerous side reactions, low binding efficiency, high cost, and unsuitability for industrial production.

[0008] A second objective of this invention is to provide a pneumococcal polysaccharide-protein CDAP conjugate.

[0009] A third objective of this invention is to provide the use of the above-mentioned pneumococcal polysaccharide-protein CDAP conjugate in the preparation of a vaccine for the prevention of pneumococcal infection.

[0010] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing a pneumococcal polysaccharide-protein CDAP conjugate, comprising the following steps: Pneumococcal polysaccharide and CDAP were added to triethylenediamine (DABCO) buffer for reaction, followed by the addition of a carrier protein for further reaction. Lysine was then added to quench the unreacted cyanate groups of the pneumococcal polysaccharide, and the mixture was purified to obtain the pneumococcal polysaccharide-protein CDAP conjugate. The triethylenediamine buffer solution comprises an aqueous solution of 5-50 mM triethylenediamine; the pH of the triethylenediamine buffer solution is 8.5-9.0.

[0011] As a further technical solution, the triethylenediamine buffer solution comprises an aqueous solution of 10-30 mM triethylenediamine; the pH of the triethylenediamine buffer solution is 8.6.

[0012] As a further technical solution, the pneumococcal polysaccharide is selected from one or more polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The final concentration of the pneumococcal polysaccharide added is 2-8 mg / mL.

[0013] As a further technical solution, the molar ratio of CDAP to pneumococcal polysaccharide is (0.5-2):1, preferably (1-1.5):1; The pneumococcal polysaccharide and CDAP are reacted under stirring conditions at a temperature of 2-8°C for 10-30 min, preferably 15-25 min.

[0014] As a further technical solution, the carrier protein is selected from one or more of diphtheria toxoid (DT), tetanus toxoid (TT), diphtheria toxoid mutant (CRM197), Haemophilus influenzae surface lipoprotein, pertussis toxin and hepatitis B surface antigen, preferably diphtheria toxoid mutant; The final concentration of the added carrier protein is 1-5 mg / mL.

[0015] As a further technical solution, the conditions for adding the carrier protein to carry out the reaction include: carrying out the reaction under stirring conditions; the reaction temperature is 2-8℃; the reaction pH is 8.5-9.0; and the reaction time is 2-6h, preferably 3-5h.

[0016] As a further technical solution, the final concentration of lysine added is 10-50 mM; And / or, the quenching reaction is carried out under stirring for 30-60 minutes.

[0017] As a further technical solution, purification includes gel filtration chromatography or tangential flow ultrafiltration; The gel filtration chromatography used Sepharose CL-4B chromatography medium, and the elution buffer was PBS buffer with a pH of 7.2-7.4 and a concentration of 10-20 mM. The tangential flow ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.1-0.3 MPa.

[0018] As a further technical solution, the average molecular weight of the pneumococcal polysaccharide-protein CDAP conjugate is 1000-5000 kDa, preferably 2000-3000 kDa.

[0019] Secondly, the present invention provides a pneumococcal polysaccharide-protein CDAP conjugate, which is prepared by the above-described preparation method.

[0020] Thirdly, the present invention provides the application of the pneumococcal polysaccharide-protein CDAP conjugate prepared by the above preparation method in the preparation of a vaccine for preventing pneumococcal infection.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to apply triethylenediamine (DABCO) as a buffer system to the CDAP binding reaction of pneumococcal polysaccharide-protein. The pKa value of DABCO is about 8.7, which is highly matched with the optimal pH range (8.5-9.0) for CDAP activation reaction. It can accurately maintain the pH stability of the reaction system, avoid the decrease in activation efficiency caused by pH drift, significantly improve the batch-to-batch consistency of the binding reaction, and solve the core pain point of poor pH stability of traditional buffer systems. 2. DABCO has a bicyclic tertiary amine structure and no hydroxyl, primary or secondary amino groups in its molecule. It will not compete with CDAP or activated polysaccharides for reaction, effectively reducing the generation of by-products and improving batch-to-batch consistency of the conjugate. 3. DABCO buffer is inexpensive, has no potential toxicity, and is simple to prepare. It can be obtained by simple dissolution and pH adjustment, making it suitable for large-scale industrial production. Compared with HEPES buffer, it can reduce production costs by more than 30%. 4. The preparation method of the present invention is compatible with polysaccharides of various serotypes of pneumococcus and mainstream carrier proteins. The conjugate has high immunogenicity and good stability. After freeze-drying, it can be stably stored at -20°C for more than 18 months. It can also effectively avoid the destruction of polysaccharide antigen epitopes and the decrease in protein immunogenicity, and is suitable for the preparation requirements of multivalent pneumococcal conjugate vaccines. 5. The preparation method of the present invention has simple steps, mild reaction conditions, and is easy to control. It does not require complex equipment and can be directly connected to the existing pneumococcal conjugate vaccine production line, showing good prospects for industrial application. Detailed Implementation

[0022] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0023] In a first aspect, the present invention provides a method for preparing a pneumococcal polysaccharide-protein CDAP conjugate, comprising the following steps: Pneumococcal polysaccharide and CDAP were added to triethylenediamine buffer for reaction, followed by the addition of a carrier protein for further reaction. Lysine was then added to quench the unreacted cyanate groups of the pneumococcal polysaccharide, and the mixture was purified to obtain the pneumococcal polysaccharide-protein CDAP conjugate. The triethylenediamine buffer solution comprises an aqueous solution of 5-50 mM triethylenediamine; the pH of the triethylenediamine buffer solution is 8.5-9.0.

[0024] The preparation method provided by this invention uses DABCO as a buffer system, which can accurately maintain the optimal pH range for CDAP activation reaction, reduce side reactions, improve the yield, purity and batch-to-batch consistency of the conjugate, and has low cost and simple process, making it suitable for large-scale industrial production.

[0025] In some optional embodiments, the triethylenediamine buffer comprises an aqueous solution of 10-30 mM triethylenediamine; the pH of the triethylenediamine buffer is 8.6. At this pH value, the DABCO buffering capacity is optimal, and the CDAP activation efficiency is maximized. The buffer can be stably stored at 4°C to prevent polymerization degradation.

[0026] In some optional embodiments, the pneumococcal polysaccharide is selected from one or more polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F; preferably, a combination of 13-valent, 15-valent, or 20-valent pneumococcal polysaccharides.

[0027] The final concentration of the pneumococcal polysaccharide added is 2-8 mg / mL, for example, but not limited to 2 mg / mL, 5 mg / mL or 8 mg / mL.

[0028] In some optional embodiments, the molar ratio of CDAP to pneumococcal polysaccharide is (0.5-2):1, preferably (1-1.5):1; The pneumococcal polysaccharide and CDAP are reacted under stirring conditions at a temperature of 2-8°C for 10-30 min, preferably 15-25 min.

[0029] Further optimization and adjustment of the molar ratio of CDAP to pneumococcal polysaccharide and the reaction time were conducted to ensure full activation of the polysaccharide while reducing side reactions.

[0030] In some optional embodiments, the carrier protein is selected from one or more of diphtheria toxoid, tetanus toxoid, diphtheria toxoid mutant, Haemophilus influenzae surface lipoprotein, pertussis toxin and hepatitis B surface antigen, preferably a diphtheria toxoid mutant; The final concentration of the added carrier protein is 1-5 mg / mL, for example, but not limited to 1 mg / mL, 3 mg / mL or 5 mg / mL.

[0031] In some optional embodiments, the conditions for adding the carrier protein to carry out the reaction include: the reaction is carried out under stirring; the reaction temperature is 2-8°C; the reaction pH is 8.5-9.0, preferably 8.6 (the pH can be maintained by adding HCl or NaOH); and the reaction time is 2-6 hours, preferably 3-5 hours. This reaction time ensures that the activated polysaccharide and the carrier protein are fully bound, while avoiding excessive cross-linking of the conjugate, which could lead to a decrease in immunogenicity.

[0032] In some alternative embodiments, the final concentration of lysine added is 10-50 mM, for example, but not limited to 10 mM, 30 mM or 50 mM; And / or, the quenching reaction is carried out under stirring for a reaction time of 30-60 min, for example, but not limited to 30 min, 40 min or 60 min.

[0033] In some alternative implementations, purification includes gel filtration chromatography or tangential flow ultrafiltration; The gel filtration chromatography used Sepharose CL-4B chromatography medium, and the elution buffer was PBS buffer with a pH of 7.2-7.4 and a concentration of 10-20 mM. The tangential flow ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.1-0.3 MPa, preferably concentrated to a concentration of 5-10 mg / mL of pneumococcal polysaccharide-protein CDAP conjugate.

[0034] In some optional embodiments, the average molecular weight of the pneumococcal polysaccharide-protein CDAP conjugate is 1000-5000 kDa, preferably 2000-3000 kDa.

[0035] Secondly, the present invention provides a pneumococcal polysaccharide-protein CDAP conjugate, which is prepared by the above-described preparation method.

[0036] This pneumococcal polysaccharide-protein CDAP conjugate can be used in the preparation of vaccines to prevent diseases caused by Streptococcus pneumoniae.

[0037] Thirdly, the present invention provides the application of the pneumococcal polysaccharide-protein CDAP conjugate prepared by the above preparation method in the preparation of a vaccine for preventing pneumococcal infection.

[0038] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0039] Example 1: Preparation of 13-valent pneumococcal polysaccharide-CRM197 CDAP conjugate (DABCO buffer system) 1. Prepare DABCO buffer solution: Weigh 0.112g of triethylenediamine (DABCO), dissolve it in 100mL of deionized water, adjust the pH value to 8.6 with 0.1mol / L HCl, prepare a 10mM DABCO buffer solution, mix well and store at 4℃ for later use. 2. Polysaccharide dissolution: Polysaccharides of Streptococcus pneumoniae types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F were selected and dissolved in the DABCO buffer solution prepared in step 1 to prepare polysaccharide solutions with a concentration of 5 mg / mL for each serum type. 3. CDAP-activated polysaccharide: CDAP was added to each serum polysaccharide solution at a molar ratio of 1:1. The mixture was reacted at 4°C and a stirring speed of 100 r / min for 1 to 3 min to obtain activated polysaccharide solutions for each serum type. 4. Polysaccharide-protein binding: Mix the activated polysaccharide solutions of each serotype, add CRM197 carrier protein, adjust the concentration of CRM197 in the system to 3 mg / mL, and the mass ratio of total pneumococcal polysaccharide to CRM197 to 1:1. Maintain the temperature at 4℃ and pH at 8.6, and stir for 4 hours. During this period, the pH is maintained by adding 0.1 mol / L HCl or NaOH dropwise to obtain the binding reaction solution. 5. Quenching reaction: Add lysine solution to the binding reaction solution, with a final lysine concentration of 30mM, and stir for 45min to quench unreacted CDAP active groups. 6. Purification: The quenched binding reaction solution was purified by Sepharose CL-4B gel filtration chromatography with PBS buffer at pH 7.3 and a concentration of 15 mM as the eluent. The solution of the conjugate corresponding to the elution peak was collected. The solution was then concentrated by tangential flow ultrafiltration membrane with a molecular weight cutoff of 100 kDa at an ultrafiltration pressure of 0.2 MPa until the conjugate concentration was 500 μg / mL.

[0040] The yield of the conjugate prepared in this embodiment was 40%~60% (the yield range of different Streptococcus pneumoniae polysaccharide CRM197CDAP conjugates, the same below), the weight average molecular weight of the conjugate was 1000~3000kDa, and it could be filtered through a 0.2μm filter membrane with a yield of 85% after filtration. Immunogenicity test showed that the conjugate could induce mice to produce high-titer specific antibodies, and the antibody titer was not inferior to that of conjugates prepared by traditional buffer systems.

[0041] Example 2: Preparation of 20-valent pneumococcal polysaccharide-TT CDAP conjugate (DABCO buffer system) 1. Prepare DABCO buffer solution: Weigh 0.224g of triethylenediamine (DABCO), dissolve it in 100mL of deionized water, adjust the pH to 8.8 with 0.1mol / L NaOH, prepare a 20mM DABCO buffer solution, mix well and store at 4℃ for later use. 2. Polysaccharide dissolution: Polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 19A, 19F, and 23F were selected and dissolved in the DABCO buffer solution prepared in step 1, and the concentration of each polysaccharide solution was adjusted to 6 mg / mL. 3. CDAP-activated polysaccharide: CDAP was added to each serum polysaccharide solution at a molar ratio of 1.2:1. The mixture was reacted at 2°C and a stirring speed of 80 r / min for 1 to 3 min to obtain activated polysaccharide solutions for each serum type. 4. Polysaccharide-protein binding: Mix the activated polysaccharide solutions of each serotype, add tetanus toxoid (TT) carrier protein, adjust the TT concentration in the system to 4 mg / mL, and the mass ratio of total pneumococcal polysaccharide to TT to 1.5:1. Maintain the temperature at 2℃ and the pH at 8.8, and stir the reaction for 5 h. During this period, the pH is maintained by adding 0.1 mol / L HCl or NaOH dropwise to obtain the binding reaction solution. 5. Quenching reaction: Add lysine solution to the binding reaction solution, with a final lysine concentration of 40mM, stir the reaction for 30min to quench unreacted CDAP active groups. 6. Purification: The quenched binding reaction solution was purified by tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.25 MPa. The mixture was concentrated to a concentration of 500 μg / mL. During the process, the mixture was washed three times with PBS buffer at pH 7.4 and a concentration of 20 mM to remove free polysaccharides and byproducts.

[0042] The yield of the conjugate prepared in this embodiment was 40% to 60%, the weight average molecular weight of the conjugate was 1000 to 5000 kDa, and it could be filtered through a 0.2 μm filter membrane with a yield greater than 80% after filtration. The stability test showed that the purity and immunogenicity of the conjugate did not decrease significantly after being stored at 2 to 8 °C for 12 months.

[0043] Example 3 1. Prepare DABCO buffer solution: Weigh triethylenediamine (DABCO), dissolve it in 100mL of deionized water, adjust the pH to 8.8 with 0.1mol / L NaOH, prepare a 5mM DABCO buffer solution, mix well and store at 4℃ for later use. 2. Polysaccharide dissolution: Polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 19A, 19F, and 23F were selected and dissolved in the DABCO buffer solution prepared in step 1, and the concentration of each polysaccharide solution was adjusted to 2 mg / mL. 3. CDAP-activated polysaccharide: CDAP was added to each serum polysaccharide solution at a molar ratio of 0.5:1. The mixture was reacted at 2°C and a stirring speed of 80 r / min for 1 to 3 min to obtain activated polysaccharide solutions for each serum type. 4. Polysaccharide-protein binding: Mix the activated polysaccharide solutions of each serum type, add tetanus toxoid (TT) carrier protein, adjust the TT concentration in the system to 1 mg / mL, maintain the temperature at 2℃ and pH at 8.8, stir the reaction for 2 h, and maintain the pH stability by adding 0.1 mol / L HCl or NaOH dropwise to obtain the binding reaction solution; 5. Quenching reaction: Add lysine solution to the binding reaction solution, with a final lysine concentration of 10 mM, stir the reaction for 30 min to quench the unreacted CDAP active groups. 6. Purification: The quenched binding reaction solution was purified by tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.25 MPa. The mixture was concentrated to a concentration of 500 μg / mL. During the process, the mixture was washed three times with PBS buffer at pH 7.4 and a concentration of 20 mM to remove free polysaccharides and byproducts.

[0044] Testing showed that the yield of the conjugate prepared in this embodiment was 30%~50%, the weight average molecular weight of the conjugate was 1000~4000kDa, and it could be filtered through a 0.2μm filter membrane with a yield greater than 80% after filtration.

[0045] Example 4 1. Prepare DABCO buffer solution: Weigh triethylenediamine (DABCO), dissolve it in 100mL of deionized water, adjust the pH to 8.8 with 0.1mol / L NaOH, prepare a 50mM DABCO buffer solution, mix well and store at 4℃ for later use. 2. Polysaccharide dissolution: Polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 19A, 19F, and 23F were selected and dissolved in the DABCO buffer solution prepared in step 1, and the concentration of each polysaccharide solution was adjusted to 8 mg / mL. 3. CDAP-activated polysaccharide: CDAP was added to each serum polysaccharide solution at a molar ratio of 2:1. The mixture was reacted at 2°C and a stirring speed of 80 r / min for 1 to 3 min to obtain activated polysaccharide solutions for each serum type. 4. Polysaccharide-protein binding: Mix the activated polysaccharide solutions of each serum type, add tetanus toxoid (TT) carrier protein, adjust the TT concentration in the system to 5 mg / mL, maintain the temperature at 2℃ and pH at 8.8, stir the reaction for 6 h, and maintain pH stability by adding 0.1 mol / L HCl or NaOH dropwise to obtain the binding reaction solution; 5. Quenching reaction: Add lysine solution to the binding reaction solution, with a final lysine concentration of 50mM, stir for 30min to quench unreacted CDAP active groups. 6. Purification: The quenched binding reaction solution was purified by tangential flow ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.25 MPa. The mixture was concentrated to a concentration of 500 μg / mL. During the process, the mixture was washed three times with PBS buffer at pH 7.4 and a concentration of 20 mM to remove free polysaccharides and byproducts.

[0046] Testing showed that the yield of the conjugate prepared in this embodiment was 40%~60%, the weight average molecular weight of the conjugate was 2000~5000kDa, it could be filtered through a 0.2μm filter membrane, the yield after filtration was greater than 80%, the free polysaccharide was less than 15%, and the polysaccharide-to-protein ratio was 0.8~2.0.

[0047] Comparative Example 1: Preparation of 13-valent pneumococcal polysaccharide-CRM197 CDAP conjugate (aqueous system) The difference from Example 1 is that the DABCO buffer solution is replaced with water.

[0048] The comparative sample yielded 20%–50% of the conjugates, with a weight-average molecular weight of 800–5000 kDa. It could pass through a 0.2 μm filter membrane, yielding 70%–80% after filtration. The pH value drifted between 0.3 and 0.5 during the reaction, indicating poor batch-to-batch consistency. Free polysaccharides showed significant differences, and the polysaccharide-to-protein ratio was relatively dispersed between 0.3 and 3.0. Immunogenicity testing revealed that the antibody titer was lower than that of the conjugate prepared in Example 1.

[0049] Comparative Example 2 The difference from Example 1 is that the DABCO buffer is replaced with borate buffer (0.2 g / L sodium tetraborate solution (pH 8.6)).

[0050] Testing revealed that the yield of the conjugate prepared in this comparative ratio was 20%–50%, the weight-average molecular weight of the conjugate was 1000–5000 kDa, and it could be filtered through a 0.2 μm filter membrane with a yield of 70%–80% after filtration. The pH value drifted within the range of 0.3–0.5 during the reaction, indicating poor batch-to-batch consistency. The free polysaccharide variation was significant, below 30%, and the polysaccharide-to-protein ratio was relatively dispersed within the range of 0.3–3.0.

[0051] By comparing Examples 1 and 2 with the comparative examples, it can be seen that the present invention uses DABCO as a buffer system, which can significantly improve the yield and purity of pneumococcal polysaccharide-protein CDAP conjugate, improve batch-to-batch consistency of the reaction, and enhance the immunogenicity and stability of the conjugate, thus having obvious technical advantages.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a pneumococcal polysaccharide-protein CDAP conjugate, characterized in that, Includes the following steps: Pneumococcal polysaccharide and CDAP were added to triethylenediamine buffer for reaction, followed by the addition of a carrier protein for further reaction. Lysine was then added to quench the unreacted cyanate groups of the pneumococcal polysaccharide, and the mixture was purified to obtain the pneumococcal polysaccharide-protein CDAP conjugate. The triethylenediamine buffer solution comprises an aqueous solution of 5-50 mM triethylenediamine; the pH of the triethylenediamine buffer solution is 8.5-9.

0.

2. The preparation method according to claim 1, characterized in that, The triethylenediamine buffer solution comprises an aqueous solution of 10-30 mM triethylenediamine; the pH of the triethylenediamine buffer solution is 8.

6.

3. The preparation method according to claim 1, characterized in that, The pneumococcal polysaccharide is selected from one or more polysaccharides of Streptococcus pneumoniae types 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. The final concentration of the pneumococcal polysaccharide added is 2-8 mg / mL.

4. The preparation method according to claim 1, characterized in that, The molar ratio of CDAP to pneumococcal polysaccharide is (0.5-2):1, preferably (1-1.5):1; The pneumococcal polysaccharide and CDAP are reacted under stirring conditions at a temperature of 2-8°C for 10-30 min, preferably 15-25 min.

5. The preparation method according to claim 1, characterized in that, The carrier protein is selected from one or more of diphtheria toxoid, tetanus toxoid, diphtheria toxoid mutant, Haemophilus influenzae surface lipoprotein, pertussis toxin and hepatitis B surface antigen, preferably a diphtheria toxoid mutant; The final concentration of the carrier protein added is 1-5 mg / mL; The conditions for adding the carrier protein to carry out the reaction include: the reaction is carried out under stirring; the reaction temperature is 2-8℃; the reaction pH is 8.5-9.0; and the reaction time is 2-6h, preferably 3-5h.

6. The preparation method according to claim 1, characterized in that, The final concentration of lysine added is 10-50 mM; And / or, the quenching reaction is carried out under stirring for 30-60 minutes.

7. The preparation method according to claim 1, characterized in that, Purification includes gel filtration chromatography or tangential flow ultrafiltration; The gel filtration chromatography used Sepharose CL-4B chromatography medium, and the elution buffer was PBS buffer with a pH of 7.2-7.4 and a concentration of 10-20 mM. The tangential flow ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 100 kDa and an ultrafiltration pressure of 0.1-0.3 MPa.

8. The preparation method according to claim 1, characterized in that, The average molecular weight of the pneumococcal polysaccharide-protein CDAP conjugate is 1000-5000 kDa, preferably 2000-3000 kDa.

9. A pneumococcal polysaccharide-protein CDAP conjugate, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of the pneumococcal polysaccharide-protein CDAP conjugate prepared by any one of claims 1-8 in the preparation of a vaccine for the prevention of pneumococcal infection.