CN108144052A - Streptococcus pneumoniae polysaccharides-protein conjugate and its preparation method and purposes - Google Patents

Abstract

An immunogenic polysaccharide-protein conjugate, which is composed of high-pressure homogenized Streptococcus pneumoniae capsular polysaccharide and protein, has immunogenicity, and can be used for the preparation of Streptococcus pneumoniae conjugate vaccine. The method for preparing immunogenic polysaccharide-protein conjugates provided by the present invention uses a physical degradation method to degrade the purified capsular polysaccharide, so that the solubility of the polysaccharide reactants participating in covalent bonding increases, the solution viscosity decreases, and avoids The occurrence of the gel phenomenon in the reaction is beneficial to the progress of the reaction and facilitates the purification of the polysaccharide-protein conjugate.

Description

Streptococcus pneumoniae polysaccharide-protein conjugate and its preparation method and use

technical field

The present invention relates to a covalent conjugate, in particular to a conjugate obtained by covalently combining polysaccharides and proteins, a method for preparing the conjugate, and a method for preparing a Streptococcus pneumoniae vaccine. application.

Background technique

Streptococcus pneumoniae (Streptococcus pneumoniae) is a spherical Gram-positive bacterium under the genus Streptococcus, which has d-hemolytic properties. Pneumonia caused by Streptococcus pneumoniae occurs most often in the very young or the elderly. Streptococcus pneumoniae is also a common cause of bacterial meningitis in adults. In addition, Streptococcus pneumoniae can also cause different kinds of diseases, such as: acute sinusitis, otitis media, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis and cellulitis, etc.

B-lactam antibiotics are mostly used in the treatment of pneumococcal disease. In the early days, almost all strains of Streptococcus pneumoniae were sensitive to penicillin. With the increase in the use of antibiotics, the resistance of bacteria to antibiotics also increased.

Since 2000, a seven-valent Streptococcus pneumoniae conjugate vaccine has been recommended in the United States, mainly suitable for infants aged 2 months to 23 months or children aged 2 years to 5 years, which can protect children from Streptococcus pneumoniae deep infection, such as: septicemia and meningitis.

Streptococcus pneumoniae produces capsules in humans and animals. According to the antigenicity of polysaccharides in the capsule, pneumococcus has been divided into 91 serotypes.

Chinese invention patent application 200680017776.8 discloses a polyvalent pneumococcal polysaccharide-protein conjugate composition, which contains 13 different polysaccharide-protein conjugates, and a physiologically acceptable carrier, wherein each conjugate contains Capsular polysaccharides from different serotypes of Streptococcus pneumoniae conjugated to carrier proteins, and the capsular polysaccharides from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F were prepared, wherein the carrier protein was CRM197.

The relative molecular mass of the capsular polysaccharide of Streptococcus pneumoniae is relatively large, usually in the range of 1,000KD to 3,000KD. For immunogenic substances containing polysaccharides, the value of the relative molecular mass of polysaccharides is an important indicator to ensure antigenicity. The greater the relative molecular mass, the stronger the antigenicity, and the reduction of relative molecular mass may be accompanied by the loss of antigenicity. However, for polysaccharide-protein conjugates, there are many technical problems in the process of combining huge polysaccharide molecules with protein carriers, such as: the solubility of polysaccharides is too low, the viscosity of polysaccharide solutions is too high, and cross-linking is easy to occur during the binding reaction. Gel formation, complex conjugates and difficult purification, etc., resulting in a large residual amount of reactants in the conjugate, high content of free polysaccharides, low reaction yield, and the best immunogenicity of the conjugate It is closely related to the molecular size of the polysaccharide fragment in the conjugate.

Contents of the invention

An object of the present invention is to provide a polysaccharide-protein conjugate with immunogenicity, which is convenient for preparation and isolation of the conjugate, and the immunogenicity is also preserved.

Another object of the present invention is to provide a method for preparing polysaccharide-protein conjugates with immunogenicity, optimize the binding process of capsular polysaccharides and carrier proteins, improve the binding efficiency and yield of conjugates, and improve the efficiency of conjugation. The stability and immunogenicity of the drug stock solution are convenient for subsequent purification and application.

Another object of the present invention is to provide an application of an immunogenic polysaccharide-protein conjugate in the preparation of a Streptococcus pneumoniae conjugate vaccine.

An immunogenic polysaccharide-protein conjugate derived from Streptococcus pneumoniae (serotypes such as: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A , 12F, 14, 15B, 16F, 17F, 18C, 19A, 19F, 20, 20F, 23A, 23F and 33F), the capsular polysaccharides have a molecular weight of 150,000Da to 400,000Da (especially 200,000Da to 300,000 Da) Conjugates with proteins.

Another polysaccharide-protein conjugate with immunogenicity is a conjugate composed of capsular polysaccharide from 18C serotype Streptococcus pneumoniae with a molecular weight of 150,000Da-400,000Da and CRM197 protein.

Another polysaccharide-protein conjugate with immunogenicity is a conjugate composed of capsular polysaccharide from 18C serotype Streptococcus pneumoniae with a molecular weight of 200,000Da-300,000Da and CRM197 protein.

A method for preparing immunogenic polysaccharide-protein conjugates. Firstly, the capsular polysaccharide of Streptococcus pneumoniae is degraded by physical shearing, and then enriched by ultrafiltration to obtain the degraded capsular polysaccharide. Activate the degraded capsular polysaccharide, adjust the pH value, then mix the activated degraded capsular polysaccharide and CRM197 protein, at room temperature, let the degraded capsular polysaccharide interact with CRM197 protein for 30 minutes to 60 minutes, and then stop adding After the reaction system is kept overnight (such as: 16 hours to 24 hours) at a low temperature (such as: 2°C to 8°C), it is filtered with a clarification filter and concentrated and dialyzed with an ultrafiltration membrane to obtain a monovalent pneumococcal polysaccharide and protein conjugate. stock solution.

Physical shear methods such as: emulsification and homogenization (eg: high pressure homogenization). Under the action of physical shearing, the macromolecules in the solution are crushed and broken under the action of strong shearing, impact and cavitation, resulting in their degradation, but their structural units remain basically unchanged.

The function of homogeneity is to uniformly disperse different components in the same system, break up or refine the insoluble phase particles in the liquid, and form a stable and uniform solution.

The material enters the homogenizing valve under the push of the plunger, and is pressurized and discharged in the homogenizing valve. During this process, the material is subjected to various effects: cavitation, high-frequency vibration of strong turbulent flow, shearing action with the edge of the homogeneous valve, and impact with the homogeneous valve stop ring.

Homogenization can be divided into: bead milling method (belonging to solid shearing, which can reach a higher crushing rate and can be operated on a large scale, and the target product of macromolecules is easily inactivated, and slurry separation is difficult), high-pressure homogenization method (belonging to liquid shearing cutting effect, can achieve high crushing rate, can be operated on a large scale, for a small amount of material <100ml, difficult to operate), ultrasonic crushing method (belongs to liquid shearing effect, the effect on yeast is poor, the temperature rises violently during the crushing process, and is not suitable for large-scale Scale operation), and X-press method (belonging to solid shearing, high fragmentation rate, high activity retention rate, not suitable for freeze-sensitive target products).

Activators such as: 1-cyano-4-dimethylamino-pyridine tetrafluoroboric acid, cyanogen bromide and sodium periodate. These compounds are used in the present invention individually and in combination. The dosage is 50w/w%～100w/w% of the weight of the polysaccharide (i.e. degraded capsular polysaccharide), such as: but not limited to 50w/w%, 55w/w%, 60w/w%, 65w/w%, 70w/ w%, 75w/w%, 80w/w%, 85w/w%, 90w/w%, 95w/w% and 100w/w%, especially 65w/w%～80w/w%.

The pH of the interaction between the capsular polysaccharide and the CRM197 protein is 8.90-9.60, the temperature is room temperature, and the time is 3-5 minutes.

It has been verified that the prepared polysaccharide-protein conjugate has immunogenicity, and then adding excipients and auxiliary agents to prepare a vaccine for the prevention and treatment of Streptococcus pneumoniae.

The beneficial effect that technical solution of the present invention realizes:

The invention adopts a physical degradation method to degrade the purified capsular polysaccharide, and has the characteristics of simple operation, high degradation efficiency and good controllability. The sheared polysaccharide has concentrated relative molecular weight distribution, can be scaled up linearly, and is suitable for large-scale production.

The present invention uses a physical degradation method to degrade the purified capsular polysaccharide, so that the solubility of the polysaccharide reactants participating in covalent bonding increases, the viscosity of the solution decreases, avoids the occurrence of gel phenomenon in the reaction, facilitates the reaction, and facilitates the polysaccharide- Protein conjugates were purified.

Description of drawings

Fig. 1 is the ELISA detection result of the degraded polysaccharide-protein conjugate of the present invention;

Fig. 2 is the ELISA detection result of the undegraded 18C polysaccharide-protein conjugate of the present invention.

Detailed ways

The technical solution of the present invention will be described in detail below in conjunction with the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the invention can be modified or equivalently replaced , without departing from the spirit and scope of the technical solution of the present invention, all of which shall be covered by the claims of the present invention.

Example 1

In this example, by comparing the control standard with the test product, more than one serotype of pneumococcal capsular polysaccharides are qualitatively characterized. For example: Table 1 lists the identification reference information of several serotypes of capsular polysaccharides. Confirm the characteristic area of the hydrogen spectrum, integrate the obtained spectrum, and compare it with the reference standard and the test product for qualitative.

Reagents used: 1) heavy water: sigma heavy water, purity 99%; 2) standard polysaccharide: purchased from Danish Serum Institute (SSI).

Determination method: Take 5 mg of standard polysaccharide of the serotype to be tested, add 1 ml of heavy water, mix well after reconstitution, and lyophilize overnight. Add 1ml of heavy water again to redissolve and mix thoroughly to obtain a 5mg/ml standard polysaccharide solution. Measure 600 μl of the sample and place it in a nuclear magnetic resonance tube for 600M nuclear magnetic resonance measurement. Another test sample was taken and determined in the same way.

Table 1

Example 2

The most prevalent serotype 18C pneumococcus in China was selected, and the degraded capsular polysaccharide was obtained by physically shearing the 18C capsular polysaccharide.

This embodiment uses an ATS homogenizer, which has the characteristics of high crushing efficiency, large processing capacity, instant crushing, material residence time of less than 1 second, and in-situ cooling (to ensure that the discharge temperature is lower than 10 degrees).

Separation and purification on a Sephacry1S-300 (XK-50) column, and collection in sections yielded several polysaccharide chains with molecular weights ranging from 200,000Da to 300,000Da. The molecular weight was determined by high-pressure liquid phase technology, so as to optimize the physical shearing process of macromolecular polysaccharides.

Example 3

Purified capsular polysaccharide (undegraded) and capsular polysaccharide degraded by physical shear were activated by 1-cyano-4-dimethylamino-pyridine tetrafluoroboric acid (CDAP) as a polysaccharide activator, and coupled with CRM197 Combined (see FEBS Letters, 1983, 154, 209-210), the polysaccharide-CRM197 protein conjugate was prepared.

The specific method is: undegraded 18C capsular polysaccharide and degraded capsular polysaccharide solution (5mg/ml～10mg/ml) are mixed with CDAP solution (the amount of CDAP is 50w/w%～100w/w% of the total amount of polysaccharide) , adjust the pH to 8.90-9.60, activate at room temperature for 1-5 minutes. Then add CRM197 protein and react at room temperature for 30 minutes to 60 minutes; then add excess glycine to terminate the reaction system and keep it at room temperature for 30 minutes. Finally, the reactant is placed at 2° C. to 8° C. for 16 hours to 24 hours.

Sampling was taken in the middle to detect the KD value (a constant characterizing the binding rate of polysaccharides and proteins), and after the reaction was completed, the reaction conjugate was clarified, concentrated and ultrafiltration dialysis to remove free protein and reaction reagents.

For the undegraded 18C capsular polysaccharide and the degraded capsular polysaccharide, its binding ratio to protein is shown in Table 2.

The sugar/protein ratio (0.3-3.0), binding reaction yield (more than 20%) and antigenicity of the conjugate were analyzed (see Examples 4 and 5 for details).

Table 2

For the data in Table 2, the KD value of the conjugate was detected by HPLC, the total polysaccharide content and free polysaccharide content in the stock solution were detected by the anthrone method, the total protein content in the stock solution was detected by the Lowry method, and the free protein content and residual reagents were measured by HPLC Wait.

Example 4

The ELISA method was used to detect the influence of polysaccharide fragments of different sizes on the immunogenicity of the polysaccharide-protein conjugate of Streptococcus pneumoniae type 18C, and the test method was as follows:

(1) Standard polysaccharide-coated ELISA plate, 100 μl per well, placed at 4°C for overnight coating. (2) After coating, take out the ELISA plate, discard the coating solution, wash the plate three times with PBST, add 150 μl of blocking solution to block, and incubate at 37°C for 60 min. (3) After blocking, take out the ELISA plate and discard the blocking solution. (4) Add immune serum, 100 μl per well, and incubate at 37° C. for 90 minutes. (5) Wash the plate, the method is the same as step (3). (6) Add goat anti-mouse HRP-IgG, 100 μl per well, and incubate at 37° C. for 1 hour. (7) Wash the plate, the method is the same as step (3). (8) Add TMB, 100 μl per well, and place in the dark for 4.5 minutes. (9) Add stop solution to stop the reaction. (10) Read the enzyme label at 450nm-620nm.

See Table 3 and its corresponding Figure 1 for the test results.

Table 3 ELISA detection results of polysaccharide-protein conjugates after degradation

Example 5

The purified polysaccharide-protein conjugates were used to immunize experimental mice and samples were taken. The experimental process was as follows:

The stock solution of 18C polysaccharide-CRM conjugate was injected intraperitoneally into NIH mice (female: 10g-12g, 30 mice) at week 0, week 2 and week 4 respectively, each injection dose was 0.5ml (about 2.2μg conjugated 10 mice were collected at the 2nd week, 4th week and 6th week respectively, eyeball blood was collected, serum was separated by centrifugation, and stored at no higher than -20°C.

See Table 4 and its corresponding Figure 2 for test results.

Table 4 ELISA detection results of undegraded 18C polysaccharide-protein conjugates

According to the data obtained in Example 4 and Example 5 (Table 3 and Table 4, and corresponding Figure 1 and Figure 2), it can be seen that the antibody titer of the sample increased significantly after each immunization, and the antibody titer increased most obviously in the sixth week.

Claims (14)

1. a kind of polysaccharide-protein conjugate with immunogenicity, it is characterised in that by molecular weight be 150,000Da~400, The conjugate of the polysaccharide of 000Da and protein composition, the polysaccharide come from streptococcus pneumoniae capsular polysaccharide.

2. the polysaccharide-protein conjugate according to claim 1 with immunogenicity, it is characterised in that the polysaccharide By the streptococcus pneumoniae capsular polysaccharide being emulsified or high-pressure homogeneous method is produced.

3. the polysaccharide-protein conjugate according to claim 1 with immunogenicity, it is characterised in that the albumen Matter is CRM197 albumen.

4. the polysaccharide-protein conjugate according to claim 1 with immunogenicity, it is characterised in that the polysaccharide Molecular weight be 200,000Da~300,000Da.

5. the polysaccharide-protein conjugate according to claim 1 with immunogenicity, it is characterised in that the pneumonia chain The capsular polysaccharide of coccus be taken from serotype for 1,2,3,4,5,6A, 6B, 7F, 8,9N, 9V, 10A, 11A, 12F, 14,15B, 16F, 17F, 18C, 19A, 19F, 20, the streptococcus pneumonias of the one or more of 20F, 23A, 23F and 33F.

A kind of 6. method for producing the polysaccharide-protein conjugate described in claim 1 with immunogenicity, it is characterised in that First streptococcus pneumoniae capsular polysaccharide is degraded using physical shear, then ultrafiltration enrichment, obtain the capsular polysaccharide of degradation, then lead to It crosses activator to activate the capsular polysaccharide of degradation, adjusts pH value, then by the capsular polysaccharide of the degradation of activation and CRM197 eggs It mixes in vain, in room temperature, makes the 30 minutes~addition termination after sixty minutes of capsular polysaccharide and CRM197 protein-interactings of degradation Agent after reaction system is stayed overnight at low temperature, is filtered, ultrafiltration membrane concentration dialysis with clarification filter, so as to obtain pneumococal polysaccharide With protein conjugate unit price stoste.

7. according to the method described in claim 6, it is characterized in that activator is selected from 1- cyano -4- Dimethylamino-pyridines four The one or more of fluoboric acid, cyanogen bromide and sodium metaperiodate.

8. according to the method described in claim 6, it is characterized in that the activator dosage for polysaccharide total amount 50w/w%~ 100w/w%.

9. according to the method described in claim 6, it is characterized in that the pH of the polysaccharide and CRM197 protein-interactings is 8.90~9.60.

10. the according to the method described in claim 6, it is characterized in that temperature of the polysaccharide and CRM197 protein-interactings For room temperature.

11. the according to the method described in claim 6, it is characterized in that time of the polysaccharide and CRM197 protein-interactings It is 1 minute~5 minutes.

12. according to the method described in claim 6, it is characterized in that the low temperature is 2 DEG C~8 DEG C.

13. according to the method described in claim 6, it is characterized in that described is 16 hours~24 hours overnight.

14. the polysaccharide-protein conjugate according to claim 1 with immunogenicity is preparing streptococcus pneumonia combination Application in vaccine.