A method of activating bacterial capsular polysaccharides

Abstract

The present application relates to a kind of methods for activating bacterial capsule polysaccharide, belong to vaccine preparation technical field.The present application activates bacterial capsule polysaccharide using TEMPO-NCS method, it is carried out in the mixed phase reaction system formed by water and organic solvent (preferably acetonitrile or DMSO), wherein, the volume fraction of organic solvent is 10%~60% (wherein preferably 20%~40%, further preferably 30%), the rest solvent is water.The bacterial capsule polysaccharide activation method provided by the present application is suitable for a variety of bacteria such as Haemophilus influenzae type b (Hib), pneumococcus, etc., and the activated polysaccharide prepared can be used to conjugate with carrier protein to prepare polysaccharide conjugate, and the polysaccharide conjugate obtained has better immunogenicity and improved safety, and can be used to prepare polysaccharide conjugate vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for activating bacterial capsular polysaccharide, and belongs to the technical field of vaccine preparation. BACKGROUND

[0002] In the preparation of bacterial polysaccharide conjugate vaccine, the prior art mainly adopts one of the following two methods to activate the capsular polysaccharide:

[0003] (1) Cyanation method: CNBr (bromocyanogen, C≡N-Br) or CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is used, which attacks the hydroxyl group (-OH) on the polysaccharide to form an active group cyanate (-OC≡N), and then the cyanate reacts with the amino group on the carrier protein directly or through a linker to form a covalent bond. Since each repeat unit of the capsular polysaccharide usually contains multiple hydroxyl groups at different sites (for example, in the capsular polysaccharide of Haemophilus influenzae type b (Hib), there are hydroxyl groups on the 2nd, 5th carbon atoms of ribose and on the 2', 3' and 4' carbon atoms of ribitol), all of which can be activated to form cyanate, so the cyanation method has the following characteristics: the activation sites are random, resulting in uncertainty of the polysaccharide hydroxyl sites combined with the carrier protein, and certain differences in the structure of the different conjugate molecules obtained, and the activation reaction is fast (completed within a few minutes), and the activated product is easily hydrolyzed, so the process operation requires higher requirements. In addition, CNBr is highly toxic, and the intermediate product isopure bond is easily hydrolyzed, so CNBr has been gradually replaced by CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate).

[0004] (2) Oxidation method: the adjacent hydroxyl groups on the polysaccharide chain (such as the adjacent hydroxyl groups on the 2', 3' carbon atoms or the 3', 4' carbon atoms of ribitol in Hib capsular polysaccharide) are oxidized by an oxidizing agent (such as sodium periodate) to form an active aldehyde group (-CHO), and then the aldehyde group directly or through a linker combines with the free amino group on the carrier protein. The formation of aldehyde group by sodium periodate oxidation is usually accompanied by chain breakage or ring opening of monosaccharide structure, so the characteristics of sodium periodate oxidation method are: the polysaccharide molecules after chain breakage and the polysaccharide-protein conjugate molecules formed by the combination of the polysaccharide molecules with the carrier protein are usually small, while large molecular weight conjugates usually have potential better stability and better immunogenicity in children; ring opening of monosaccharide structure may destroy the antigenic epitopes, affecting the immunogenicity of the final conjugate and vaccine.

[0005] In the prior art, CN103251943B, CN108295253A, CN103599529B, etc. activate bacterial capsular polysaccharide by the above-mentioned method. In view of the shortcomings of the traditional method, patent CN104870463B and WO2023025002A1 disclose a new polysaccharide activation method, i.e. a method for activating pneumococcal capsular polysaccharide by using a nitroxyl compound-co-oxidant such as TEMPO-NCS (TEMPO: 2,2,6,6-tetramethyl-1-piperidinyloxy; NCS: N-chlorosuccinimide). However, (1) there is still no report on the use of TEMPO-NCS method for activating other bacterial (e.g. Hib) capsular polysaccharides; in addition, (2) the existing technology TEMPO-NCS activates capsular polysaccharide in an aqueous phase, although water is necessary for dissolving buffer salt and participates in the catalytic cycle of TEMPO (hydrolysis step), but excessive water can accelerate the hydrolysis of NCS to generate hypochlorous acid, increase non-selective oxidation and chloro side reactions, and possibly promote the hydration of aldehyde. Therefore, it is still necessary to further improve the process of activating bacterial capsular polysaccharide by TEMPO-NCS method to reduce the side reactions in the polysaccharide oxidation process. SUMMARY

[0006] In view of the above problems, the present application provides a method for activating bacterial capsular polysaccharide based on nitroxyl compound-co-oxidant, specifically relates to a method for selectively activating the primary hydroxyl group of bacterial capsular polysaccharide to form an active intermediate by using a nitroxyl compound-co-oxidant in a mixed phase (referred to as "mixed phase") of water and an organic solvent, and then covalently combining the active intermediate with a carrier protein to form a polysaccharide-protein conjugate.

[0007] (I) In a first aspect, the present application provides a method for activating bacterial capsular polysaccharide, characterized in that a nitroxyl compound-co-oxidant is used to activate the bacterial capsular polysaccharide, and the activation reaction is carried out in a mixed phase reaction system formed by water and an organic solution.

[0008] Preferably, the organic solvent is selected from one of the following: dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), sulfolane, N-methyl-2-pyrrolidone (NMP), hexamethylphosphoramide (HMPA), DMF (dimethylformamide) or acetonitrile. Further preferably, the organic solvent is DMSO or acetonitrile.

[0009] Preferably, in the mixed phase reaction system, the volume fraction of the organic solution is X and the volume fraction of water is 1-X relative to the total volume of the reaction system, wherein X is 10% to 60%, further preferably 20% to 40%, and more preferably 30%.

[0010] Preferably, the bacterial capsular polysaccharide is selected from the group consisting of Haemophilus influenzae (e.g., Haemophilus influenzae type b), meningococcal, Streptococcus pneumoniae, and Streptococcus group B capsular polysaccharides. Further preferably, the capsular polysaccharide is selected from the group consisting of Haemophilus influenzae type b capsular polysaccharide, meningococcal Y and W group capsular polysaccharides, Streptococcus pneumoniae 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F type capsular polysaccharides, and Streptococcus group B type Ia, Ib, II, III, IV, and V capsular polysaccharides. Still further preferably, the capsular polysaccharide is Haemophilus influenzae type b capsular polysaccharide.

[0011] Preferably, the capsular polysaccharide is a refined polysaccharide or a degraded polysaccharide. Further preferably, the capsular polysaccharide is a degraded polysaccharide, and the degradation is selected from one or more of hydrolysis, enzymatic degradation, sonication, homogenization, and microfluidization.

[0012] Preferably, the nitroxyl compound is TEMPO or a derivative thereof. Further preferably, the nitroxyl compound is selected from the group consisting of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), 2, 2, 6, 6-tetramethyl-4-(methylsulfonyloxy)-1-piperidinyloxy, 4-phosphonooxy-TEMPO, 4-oxo-TEMPO, 4-methoxy-TEMPO, 4-isothiocyanato-TEMPO, 4-(2-iodoacetylamino)-TEMPO radical, 4-hydroxy-TEMPO, 4-cyano-TEMPO, 4-carboxy-TEMPO, 4-(2-bromoacetylamino)-TEMPO, 4-amino-TEMPO, 4-acetylamino-2, 2, 6, 6-tetramethylpiperidine 1-oxyl. Still further preferably, the nitroxyl compound is TEMPO.

[0013] Preferably, the co-oxidant is a co-oxidant having the ability to selectively oxidize primary alcohols in the presence of a nitroxyl compound. The co-oxidant is a molecule bearing an N-halo group, including but not limited to N-chlorosuccinimide (NCS), N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1, 3, 5-trichloro-1, 3, 5-triazine-2, 4, 6-trione, dibromoisocyanuric acid, 1, 3, 5-tribromo-1, 3, 5-triazine-2, 4, 6-trione, diiodoisocyanuric acid, and 1, 3, 5-triiodo-1, 3, 5-triazine-2, 4, 6-trione. Further preferably, the co-oxidant is N-chlorosuccinimide.

[0014] Preferably, the nitroxyl compound is used in an amount of 0.01 eq to 0.2 eq relative to the substrate, and the co-oxidant is used in an amount of 0.1 eq to 2 eq. Further preferably, the nitroxyl compound is used in an amount of 0.05 eq to 0.1 eq, and the co-oxidant is used in an amount of 0.5 eq to 1 eq. Still further preferably, the combination of the nitroxyl compound and the co-oxidant is used in an amount of 0.05 eq TEMPO + 0.5 eq NCS, or 0.1 eq TEMPO + 0.5 eq NCS, or 0.05 eq TEMPO + 1 eq NCS, or 0.1 eq TEMPO + 1 eq NCS.

[0015] Preferably, the activation method comprises the following steps:

[0016] S1. Preparation of the activation reaction system: according to the scale of the activation reaction system, add the polysaccharide solution, buffer (such as phosphate buffer or carbonate buffer), organic solvent and water so that the final concentration of the polysaccharide in the activation reaction system is 1-10 mg / mL (preferably 5 mg / mL), the final concentration of the phosphate buffer is 5-20 mM (preferably 10 mM), the volume fraction of the organic solvent is 10%-60% (preferably 20-40%, most preferably 30%), and the balance is water;

[0017] S2. Activation reaction: dissolve TEMPO and NCS in organic solvent respectively, then add them to the activation reaction solution, mix well, control the reaction to be carried out in the dark at 5±3℃, and react for 10-20 h (preferably 16 h±1 h);

[0018] S3. Terminate the reaction, remove small molecules by dialysis, and obtain the polysaccharide activation product.

[0019] (II) In a second aspect, the present application further provides a preparation method of a polysaccharide conjugate, characterized in that the bacterial capsular polysaccharide is activated by the method of the first aspect, and then is directly or through a linker covalently conjugated with a carrier protein.

[0020] Preferably, the carrier protein is selected from one, two or more of tetanus toxoid (TT), diphtheria toxoid (DT), diphtheria toxin non-toxic variant (CRM197), group B meningococcal outer membrane protein (OMP), pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumolysin (Ply), Haemophilus influenzae D protein (PD), pertussis toxin (PT), pertussis filamentous haemagglutinin (FHA), pertussis adhesin (PRN), cholera toxin (CT), muramyl dipeptide (MDP), E. coli LT, E. coli ST, tuberculin purified protein derivative (PPD), Pseudomonas aeruginosa exotoxin A (PEA), ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), hepatitis B virus surface antigen (HBsAg), hepatitis B virus core antigen (HBcAg) and tetanus toxin C fragment (TTC). Further preferably, the carrier protein is selected from TT, DT or CRM197.

[0021] In one embodiment of the present application, the activated polysaccharide is directly linked to the carrier protein. Preferably, the activated polysaccharide is linked to the carrier protein by reductive amination using an aldehyde group of the activated polysaccharide and an amino group of the carrier protein.

[0022] In one embodiment of the present application, the activated polysaccharide is linked to the carrier protein via a linker. Preferably, the linker is a homobifunctional linker, including but not limited to malonic acid dihydrazide, succinic acid dihydrazide, glutaric acid dihydrazide, adipic acid dihydrazide, butane-1,4-diamine, pentane-1,5-diamine and hexane-1,6-diamine. Further preferably, the linker is adipic acid dihydrazide.

[0023] In one embodiment of the present application, the activated polysaccharide is first linked to a linker to form a polysaccharide-linker derivative, and the polysaccharide derivative is then linked to the carrier protein; in another embodiment of the present application, the carrier protein is first linked to a linker to form a protein-linker derivative, and the protein derivative is then linked to the activated polysaccharide.

[0024] (Three) In a third aspect, the present application further provides use of the polysaccharide activation method of the first aspect in the preparation of a polysaccharide conjugate vaccine, or use of the preparation method of the polysaccharide conjugate of the second aspect in the preparation of a polysaccharide conjugate vaccine.

[0025] (Four) In a fourth aspect, the present application further provides a polysaccharide conjugate prepared by the preparation method of the polysaccharide conjugate of the second aspect. Preferably, the polysaccharide conjugate is a Haemophilus influenzae type b polysaccharide conjugate.

[0026] Explanation of terms:

[0027] TEMPO-NCS: combination of nitroxyl compound TEMPO and co-oxidant NCS. TEMPO-NCS: combination of nitroxyl compound TEMPO and co-oxidant NCS.

[0028] Polysaccharide conjugate: conjugate formed by covalent conjugation of immunogenic polysaccharide with carrier protein; vaccine prepared therefrom is called polysaccharide conjugate vaccine.

[0029] eq (abbreviation of equivalent): equivalent, used to express the stoichiometric ratio of reactants; in this application, it is the core to reflect the molar ratio relationship of nitroxyl compound (such as TEMPO), co-oxidant (such as NCS) to substrate (such as polysaccharide primary hydroxyl group); for example, 0.1 eq means that its amount is 10% of the molar number of the substrate, 1 eq means that its amount is 100% of the molar number of the substrate, when the substrate is Hib polysaccharide: according to practice, about 0.25 mmol of primary hydroxyl group is contained in 100 mg of Hib polysaccharide, ① 0.1 eq of TEMPO (156.25 g / mol) corresponding to it is about 0.025 mmol, which is equivalent to about 3.9 mg in mass; ② 1 eq of NCS (133.53 g / mol) corresponding to it is about 0.25 mmol, which is equivalent to about 33.4 mg in mass.

[0030] The present application has the beneficial technical effects:

[0031] 1. Compared with the aqueous phase reaction system, the mixed phase reaction system of water and organic solvent used in the present application can not only ensure the reactivity of the activated reagent in the aqueous solution, but also reduce the hydrolysis and side reactions of the activated reagent in the aqueous solution, thereby improving the oxidation efficiency.

[0032] 2. Since TEMPO-NCS selectively activates the primary hydroxyl group of the capsular polysaccharide, this reaction will not cause the breakage of the polysaccharide chain, thereby avoiding the reduction of immunogenicity caused by the breakage of the polysaccharide. Moreover, in the repeating units of some bacterial capsular polysaccharides, the primary hydroxyl group is unique (for example, there is only one primary hydroxyl group in each repeating unit of Hib capsular polysaccharide molecule), so the TEMPO-NCS method can realize site-specific activation and subsequent site-specific conjugation with carrier protein. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 : Schematic diagram of TEMPO-NCS method for activating polysaccharide and forming polysaccharide conjugate.

[0034] Figure 2 : Results of TEMPO-NCS (0.05 eq TEMPO + 0.5 eq NCS) activating Hib polysaccharide in water-acetonitrile mixed phase reaction system.

[0035] Figure 3Results of TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) activation of Hib polysaccharide in water-acetonitrile mixed phase reaction system.

[0036] Figure 4 Results of TEMPO-NCS (0.05 eq TEMPO + 1 eq NCS) activation of Hib polysaccharide in water-acetonitrile mixed phase reaction system.

[0037] Figure 5 Results of TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) activation of Hib polysaccharide in water-acetonitrile mixed phase reaction system.

[0038] Figure 6 Results of TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) activation of Hib polysaccharide in water-DMSO mixed phase reaction system. DETAILED DESCRIPTION

[0039] In the following examples, Hib capsular polysaccharide was used as an example to study polysaccharide activation by TEMPO-NCS method in different reaction systems.

[0040] Example 1: TEMPO-NCS (0.05 eq TEMPO + 0.5 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile mixed phase reaction system

[0041] The operation steps of this example are as follows:

[0042] 1. Polysaccharide solution: Hib capsular polysaccharide was dissolved in 10 mM phosphate buffer (PBS) to a concentration of 20 mg / mL.

[0043] 2. Preparation of activation reaction system:

[0044] (1) Polysaccharide solution: according to the scale (volume) of the activation reaction system, 20 mg / mL Hib polysaccharide solution (in 10 mM PBS) was measured to make the final concentration of polysaccharide in the activation reaction system 5 mg / mL.

[0045] (2) Buffer: according to the scale of the activation reaction system, 200 mM pH 8.6 phosphate buffer was added to the reaction system to make the final concentration of phosphate buffer in the activation reaction system 10 mM.

[0046] (3) Organic solvent: according to the scale of the activation reaction system, organic solvent (acetonitrile) was added to the reaction system to make the volume fraction of organic solvent (i.e. the ratio of the volume of organic solvent to the total volume of the reaction system) reach the "set ratio" (0%, 10%, 20%, 30%, 40%, 60%).

[0047] (4) Water: according to the scale of the activation reaction, the rest is supplemented with water to make the volume fraction of water reach "1-set ratio" (100%, 90%, 80%, 70%, 60%, 40%).

[0048] That is, in the activation reaction solution, four components including polysaccharide, buffer, organic solvent and water are included, and the concentrations (or ratios) of them are 5 mg / mL, 10 mM, set ratio, and 100%-set ratio, respectively.

[0049] Taking a 20 mL activation reaction system (in which the volume fraction of water is 70% and the volume fraction of organic solvent is 30%) as an example, the addition amount of each component and its calculation method are shown in Table 1:

[0050] Table 1. Preparation of a 20 mL activation reaction system (in which the volume ratio of water to organic solvent is 70:30)

[0051]

[0052] 3. TEMPO-NCS activation reaction: TEMPO and NCS (0.1 eq TEMPO + 0.5 eq NCS) were dissolved in an organic solvent (acetonitrile) respectively and added to the activation reaction solution, mixed well, and the reaction was controlled at 5±3℃ in the dark for about 16 h.

[0053] 4. Terminate the reaction, remove small molecules by dialysis, and obtain different Hib polysaccharide oxides.

[0054] 5. Effect detection of TEMPO-NCS activated Hib capsular polysaccharide: for the activated polysaccharide sample obtained in Example 1, the sugar content and aldehyde group content of the activated polysaccharide were determined according to 5.1 and 5.2 below, the oxidation degree was calculated according to 5.3 below, and the carboxyl group content was determined according to 5.4 below, in order to compare the polysaccharide activation effect of TEMPO-NCS method in mixed phase reaction system and in aqueous phase reaction system, and the degree of generation of carboxyl by-product.

[0055] 5.1 Polysaccharide content detection: the polysaccharide content was determined according to the current edition of Chinese Pharmacopoeia 3421.

[0056] 5.2 Aldehyde group content detection (MBTH method): a) Prepare gradient standard solutions with aldehyde group content of 0, 15, 30, 45, 60, 75 μmol / L, 1 mL per tube, in duplicate, using glycerinaldehyde dimer as standard; add 1 mL of mixed solution of MBTH (5 mg / mL) / SA (sulfamic acid, 10 mg / mL) to each tube, shake to mix, and let stand at room temperature for 1 h; then add 1 mL of mixed solution of FAS (ferric ammonium sulfate, 15 mg / mL) / SA (10 mg / mL) to each tube, shake to mix, and let stand at room temperature for 0.5 h for color development; use the standard tube with 0 aldehyde group content as blank control, measure the absorbance of each gradient standard solution at wavelength 610 nm, and plot the standard curve with the concentration of gradient standard solution as abscissa and the absorbance as ordinate to obtain the regression equation. b) Dilute the polysaccharide oxidate sample solution to within the standard solution concentration range, take 1 mL, in duplicate; treat for color development and read at 610 nm as above, and calculate the aldehyde group content of the polysaccharide oxidate according to the regression equation and the dilution factor of the polysaccharide oxidate sample.

[0057] 5.3 Degree of oxidation calculation

[0058] The degree of oxidation (DO) is the number of polysaccharide repeat units (the smallest basic unit of which the chemical composition and structure can be repeatedly present in a polysaccharide molecule) corresponding to a unit aldehyde group, for example, the number of moles of polysaccharide repeat units corresponding to a mole of aldehyde group, i.e., DO = moles of polysaccharide repeat units / moles of aldehyde group; the moles of polysaccharide repeat units are calculated according to the polysaccharide content detection result, the moles of aldehyde group are calculated according to the aldehyde group content detection result, and the DO value is calculated according to the above formula. The smaller the DO value, the fewer the number of polysaccharide repeat units corresponding to a unit aldehyde group, i.e., the higher the proportion of aldehyde groups produced and the better the polysaccharide activation effect.

[0059] 5.4 Carboxyl group content and proportion detection (for evaluating the extent of side reactions)

[0060] If carboxyl groups are generated instead of aldehyde groups during oxidation, since carboxyl groups cannot participate in subsequent reactions to generate the final polysaccharide protein conjugate, they are undesirable byproducts, and it is necessary to detect the carboxyl group content for quality monitoring and control.

[0061] Carboxyl content detection (titration method): Take 10 mL of dialyzed or ultrafiltrated polysaccharide oxide sample, add 2-3 drops of phenolphthalein indicator, titrate with 1 mmol / L NaOH solution until the red color appears, which is the end point of titration. Record the volume of consumed NaOH solution, and calculate the number of moles of consumed NaOH, which is the number of moles of carboxyl groups in the sample. Then, calculate the percentage of carboxyl-containing polysaccharide repeating units in the total polysaccharide (referred to as carboxyl ratio). The lower the carboxyl ratio, the less the generation of carboxyl by-products.

[0062] The results of oxidation degree and by-product carboxyl ratio detection are shown in Table 2, Figure 2 .

[0063] Table 2. Results of TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile reaction system

[0064]

[0065] Example 2: TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile mixed phase reaction system

[0066] This example is carried out according to steps 1-5 of Example 1, except that the amount of TEMPO and NCS in step 3 is: 0.1 eq TEMPO + 0.5 eq NCS.

[0067] The results of oxidation degree and by-product carboxyl ratio detection are shown in Table 3, Figure 3 .

[0068] Table 3. Results of TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile reaction system

[0069]

[0070] Example 3: TEMPO-NCS (0.05 eq TEMPO + 1 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile mixed phase reaction system:

[0071] This example is carried out according to steps 1-5 of Example 1, except that the amount of TEMPO and NCS in step 3 is: 0.05 eq TEMPO + 1 eq NCS.

[0072] The results of oxidation degree and by-product carboxyl ratio detection are shown in Table 4, Figure 4 .

[0073] Table 4. Results of TEMPO-NCS (0.05 eq TEMPO + 1 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile reaction system

[0074]

[0075] Example 4: Operation steps of TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile mixed phase reaction system

[0076] This example is carried out according to steps 1-5 of Example 1, except that the amount of TEMPO and NCS used in step 3 is: 0.1 eq TEMPO + 1 eq NCS.

[0077] The results of the degree of oxidation and the proportion of byproduct carboxyl are shown in Table 5, Figure 5 .

[0078] Table 5. Results of TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) activation of Hib capsular polysaccharide in water-acetonitrile reaction system

[0079]

[0080] Tables 2-5 and Figures 2-5 show that, regardless of the amount of TEMPO and NCS used (0.05 eq TEMPO + 0.5 eq NCS, or 0.1 eq TEMPO + 0.5 eq NCS, or 0.05 eq TEMPO + 1 eq NCS, or 0.1 eq TEMPO + 1 eq NCS), the results of the degree of oxidation and the proportion of carboxyl in different reaction systems (five different proportions of mixed phase reaction system vs. aqueous reaction system) show that:

[0081] 1. With the increase of the proportion of organic solvent, the degree of oxidation level experienced a decrease and then an increase as the turning point of 70% water: 30% acetonitrile, while the proportion of carboxyl roughly showed a downward trend.

[0082] 2. Since the smaller the degree of oxidation indicates the fewer the number of polysaccharide repeating units corresponding to the unit aldehyde group (i.e. the higher the proportion of aldehyde group, indicating better polysaccharide activation effect), and the lower the proportion of carboxyl indicates the fewer the production of carboxyl byproduct, it can be seen that: 1) the effect of mixed phase reaction system is better than that of aqueous reaction system; 2) among the five different proportions of mixed phase reaction system in the horizontal comparison, the degree of oxidation obtained in the 70% water: 30% acetonitrile mixed phase reaction system is the lowest and the proportion of carboxyl is relatively low, therefore, in terms of the degree of oxidation and the proportion of carboxyl, the 70% water: 30% acetonitrile mixed phase reaction system is the best (in terms of the degree of oxidation).

[0083] Example 5: TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) in water-DMSO mixed phase reaction system to activate Hib capsular polysaccharide

[0084] This example was performed according to steps 1-5 of Example 1, except that in steps 2 and 3, DMSO was used instead of acetonitrile as the organic solvent; and in step 3, the amounts of TEMPO and NCS used were 0.1 eq TEMPO + 1 eq NCS (same as in Example 4).

[0085] The results of the degree of oxidation and the proportion of by-product carboxyl groups are shown in Table 6, Figure 6 .

[0086] Table 6. Results of TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) in water-DMSO reaction system to activate Hib capsular polysaccharide

[0087]

[0088] Table 6 and Figure 6 show that, similar to the water-acetonitrile mixed phase reaction system, the water-DMSO mixed phase reaction system also shows a decrease followed by an increase in the degree of oxidation as the proportion of organic solvent increases, with the inflection point being 70% water: 30% organic solvent, and the proportion of carboxyl groups generally decreases as the proportion of organic solvent increases.

[0089] Example 6: Preparation of Hib conjugate stock solution

[0090] As shown in Figure 1 , first, Hib activated polysaccharide (with its only primary hydroxyl group oxidized to an aldehyde group) was prepared using the TEMPO-NCS method, and then, using general conjugation techniques well known in the art, the activated polysaccharide was directly or through a linker (e.g., ADH) conjugated to a carrier protein (such as CRM197 or TT) to form a conjugate, resulting in Sample 1A and Sample 2.

[0091] Sample 1A: TEMPO-NCS (0.1 eq TEMPO + 1 eq NCS) method, Hib polysaccharide was activated in a pure water reaction system, and then the activated polysaccharide was conjugated with TT-ADH to obtain a Hib polysaccharide conjugate;

[0092] Sample 1B: TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) method, Hib polysaccharide was activated in a pure water reaction system, and then the activated polysaccharide was conjugated with TT-ADH to obtain a Hib polysaccharide conjugate

[0093] Sample 2: TEMPO-NCS (0.1 eq TEMPO + 0.5 eq NCS) method, Hib polysaccharide was activated in 70% water: 30% acetonitrile mixed phase reaction system, then the activated polysaccharide was conjugated with TT-ADH to obtain the Hib polysaccharide conjugate.

[0094] (1) TT-ADH: TT is tetanus toxoid as a carrier protein; ADH is adipic acid dihydrazide as a linker. (2) Compared with sample 1A, the amount of NCS in sample 1B is reduced to 50% of the original amount while keeping the amount of TEMPO the same; (3) Compared with sample 1B, the difference between sample 2 is that a 70% water: 30% acetonitrile mixed phase reaction system is used instead of a pure water reaction system to investigate the technical effect obtained under different reaction systems; (4) Compared with sample 1A, the difference between sample 2 is that a 70% water: 30% acetonitrile mixed phase reaction system is used on the one hand, and the use of NCS is reduced on the other hand, to investigate whether better technical effects can be achieved under the conditions of using a mixed phase reaction system and using less NCS.

[0095] Then, the main physicochemical indexes of the conjugate (including polysaccharide content, protein content, free polysaccharide, free protein and molecular size, all of which were determined by conventional experimental methods in the art) were determined, and the results are shown in Table 7.

[0096] Table 7. Physicochemical property detection results of sample 1A, sample 2 and sample 1B

[0097]

[0098] Table 7 shows that compared with sample 1A, sample 1B has a significantly reduced polysaccharide content (33.8%) and protein content (12.4%), which is due to the increase in polysaccharide oxidation degree and the decrease in conjugation efficiency. It can be seen that in a pure water reaction system, the overall reaction efficiency of polysaccharide and protein conjugation is significantly reduced, and the reduction of NCS dosage is not conducive to the activation of Hib and its conjugation with carrier protein.

[0099] Compared with sample 1A, sample 2 has a slightly increased polysaccharide content (6%) and protein content (10.1%), and overall shows a significantly better polysaccharide protein conjugation efficiency. It can be seen that although the amount of NCS is reduced, the polysaccharide activation and carrier protein conjugation efficiency do not decrease but increase under the condition of using a mixed phase reaction system. Therefore, the mixed phase reaction system brings higher polysaccharide activation efficiency, resulting in better quality of polysaccharide conjugate.

[0100] Example 7: Immunogenicity comparison

[0101] The immunogenicity of the prepared conjugates was further verified by experimental animals (NIH mouse efficacy test) for the samples 1A, 1B and 2 prepared in Example 3. The samples 1A, 1B and 2 and a positive control (ActHIB: polysaccharide conjugate vaccine prepared by conjugating Hib capsular polysaccharide activated by CDAP method with TT carrier protein, produced by Sanofi Pasteur) were used to immunize NIH mice subcutaneously on the abdomen at 1 / 4 human dose, with a total of three injections at 0 / 14 / 28 days, and blood was collected at 21 / 35 days for ELISA to determine the seroconversion rate and antibody titer.

[0102] Table 8. Immunogenicity results of samples 1A, 1B, 2 and positive control

[0103]

[0104] Table 8 shows that:

[0105] 1. The samples 1A, 1B, 2 and positive control all achieved 100% seroconversion rate after the second and third immunization.

[0106] 2. The sample 2 showed different degrees of advantage compared to sample 1A after the second and third immunization. It can be seen that, under the condition of using 70% water: 30% acetonitrile mixed phase reaction system and halving the amount of NCS (1 eq→0.5 eq), the sample 2 has significantly better immunization effect compared to the sample 1A using aqueous phase system and more NCS, reflecting that the polysaccharide protein conjugate corresponding to the sample 2 has stronger immunogenicity.

[0107] 3. The sample 2 showed obvious superior efficacy compared to sample 1B after the second and third immunization. It can be seen that, under the condition of using the same feeding ratio, the antibody titer of the mixed phase reaction system has a significant advantage compared to the pure aqueous phase system, reflecting that the polysaccharide protein conjugate corresponding to the sample 2 has stronger immunogenicity.

[0108] 4. The sample 2 obtained significantly higher GMT compared to the positive control after the second and third immunization. It can be seen that the sample 2 achieved significantly better immunostimulatory effect than the prior art.

[0109] In the above examples, the present application takes Hib as an example to prove that the TEMPO-NCS method has the advantages of high activation efficiency and less side reactions in the mixed phase reaction system, especially in the mixed phase reaction system formed by 70% water: 30% organic solvent (acetonitrile or DMSO); the activated Hib polysaccharide can be used for conjugation with carrier protein to prepare polysaccharide conjugate, and the Hib polysaccharide conjugate prepared in the mixed phase reaction system has higher immunogenicity and better safety compared to the pure water reaction system.

[0110] As can be seen from the above examples, the method for activating bacterial capsular polysaccharide based on TEMPO-NCS provided by the application has feasibility, high efficiency and safety. Since the oxidation of the primary hydroxyl group of the polysaccharide into an aldehyde group is the commonality of the TEMPO-NCS method, the application is not only suitable for the activation of Hib polysaccharide, but also suitable for the activation of capsular polysaccharides of other bacteria (including but not limited to Haemophilus influenzae, meningococcus, Streptococcus pneumoniae or group B streptococcus capsular polysaccharide, etc.).

Claims

1. A method of activating bacterial capsular polysaccharide, characterized by, The nitroxyl compound-co-oxidant is used to activate bacterial capsular polysaccharide, and the activation reaction is carried out in a mixed-phase reaction system formed by water and an organic solvent; wherein the nitroxyl compound is TEMPO, the co-oxidant is N-chlorosuccinimide, the organic solvent is acetonitrile or DMSO, and the volume fraction of the organic solvent in the mixed-phase reaction system is X, and the volume fraction of water is 1-X, wherein X is 20% to 40%.

2. The method of activating bacterial capsular polysaccharides according to claim 1, characterized in that, In the mixed-phase reaction system, the value of X is 30%.

3. The method of activating bacterial capsular polysaccharides according to claim 1, characterized in that, The amount of the nitroxyl compound is 0.01 eq to 0.2 eq, and the amount of the co-oxidant is 0.1 eq to 2 eq.

4. The method of activating bacterial capsular polysaccharides according to claim 3, characterized in that, The amount of the nitroxyl compound is 0.05 eq to 0.1 eq, and the amount of the co-oxidant is 0.5 eq to 1 eq.

5. The method of activating bacterial capsular polysaccharide according to any one of claims 1-4, characterized in that, The method comprises the following steps: S1. Preparation of the reaction system: according to the scale of the reaction system, polysaccharide solution, buffer, organic solvent and water are added so that the final concentration of the polysaccharide is 1 to 10 mg / mL, the final concentration of the buffer is 5 to 20 mM, the volume fraction of the organic solvent is 10% to 60%, and the rest is water; S2. Activation reaction: TEMPO and NCS are dissolved in the organic solvent respectively, and then added to the activation reaction solution, mixed, and controlled to carry out the reaction in the dark at 5±3℃ for 10 to 20 hours; S3. Termination of the reaction, removal of small molecules by dialysis, and obtaining of the polysaccharide activation product.

6. The method of activating bacterial capsular polysaccharides according to claim 1, characterized in that, The bacterial capsular polysaccharide is selected from the group consisting of Haemophilus influenzae, meningococcal, Streptococcus pneumoniae or group B streptococcal capsular polysaccharide.

7. A method of preparing a polysaccharide conjugate, characterized by, The bacterial capsular polysaccharide is activated by the method of any one of claims 1 to 6, and then the activated polysaccharide is directly or through a linker covalently conjugated with a carrier protein.

8. Use of the method for preparing polysaccharide conjugates according to claim 7 in the preparation of polysaccharide conjugate vaccines.

9. A polysaccharide conjugate prepared by the method according to claim 7.

10. The polysaccharide conjugate according to claim 9, characterized in that, The polysaccharide conjugate is Haemophilus influenzae type b polysaccharide conjugate. The polysaccharide conjugate is Haemophilus influenzae type b polysaccharide conjugate.

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