WO2017036142A1

WO2017036142A1 - Preparation method of group a meningococcal capsular polysaccharide conjugate vaccine

Info

Publication number: WO2017036142A1
Application number: PCT/CN2016/078089
Authority: WO
Inventors: 伍长华; 王乾; 李洪光
Original assignee: 成都欧林生物科技股份有限公司
Priority date: 2015-08-31
Filing date: 2016-03-31
Publication date: 2017-03-09
Also published as: CN105056228A

Abstract

The present invention discloses a preparation method of a group A meningococcal capsular polysaccharide conjugate vaccine. The preparation method comprises the following steps: diluting and activating group A meningococcal capsular polysaccharide, and coupling the same with sebacic dihydrazid to obtain a derivative of group A meningococcal polysaccharide and sebacic dihydrazid; and reacting, using carbodiimide as a catalyst, the derivative of group A meningococcal polysaccharide and sebacic dihydrazid with a tetanus toxoid solution, to obtain a conjugate of group A meningococcal capsular polysaccharide and a carrier protein. The present invention uses sebacic dihydrazide as a coupling agent during the preparation process of the group A meningococcal conjugate vaccine; sebacic dihydrazide has a longer molecular chain to better overcome steric hindrance between large biomolecules, thereby improving the conjugate yield.

Description

Method for preparing group A meningococcal capsular polysaccharide conjugate vaccine

Technical field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for preparing a group A meningococcal capsular polysaccharide-binding vaccine.

Background technique

[0002] Neisseria meningitidis (Nm) is the primary pathogen of meningitis and explosive sepsis. The incidence of epidemic cerebrospinal meningitis (hereinafter referred to as epidemic cerebrospinal meningitis) is often sporadic, in each case. There is no obvious correlation, usually erupting on a small scale, and in some areas it can be turned into a catastrophic, unpredictable endemic epidemic. There are about 500,000 people living with the brain each year and 50,000 deaths. Nm is an aerobic bacterium, Gram-negative, with a capsule, usually in pairs (dipococcus). Nm can be divided into 13 serogroups according to the chemical composition of the capsular polysaccharide, according to the outer membrane protein (Outer mem-bmne

Protein, OMP) The antigenic differences between PorB and POrA can be divided into different serotypes and serum subtypes. Meningococcal strains A, B, C, Y, and W135 are the major pathogenic serogroups, and there are significant geographic differences in serogroup distribution. More than 90% of cases of meningococcal disease in China are caused by Neisseria meningitidis.

[0003] Studies have shown that a group of meningococcal capsular polysaccharide vaccines are suitable for children and adults over 2 years of age. For children younger than 18 months, this PRP-based vaccine is extremely inefficient.

[0004] The development of a group of meningococcal conjugate vaccines successfully solved the problem of vaccines being ineffective in infants and young children. The combination vaccine has stronger immunogenicity and better immune effect than the previous polysaccharide vaccine, and is very effective in infants and young children. It is safe and effective even if the group A meningococcal conjugate vaccine is used in infants and young children.

[0005] In the preparation process of group A meningococcal conjugate vaccine, the currently widely used preparation methods are: preparation of group A meningococcal liquid by liquid fermentation, sterilization, followed by CDAP precipitation, ethanol precipitation, cold phenol extraction , purified ethanol and acetone washing steps to obtain purified group A capsular polysaccharide, and then activated the Hib capsular polysaccharide with CNBr, then connected with adipic hydrazide, and then under the catalysis of carbodiimide, group A cerebral capsular The polysaccharide is bound to the tetanus toxoid protein by the bridging action of adipic hydrazide, and finally the polymer polysaccharide protein conjugate is purified by gel filtration chromatography, which is a polysaccharide protein conjugate vaccine stock solution.

[0006] At present, in the preparation of group A meningococcal conjugate vaccine, group A meningococcal capsular polysaccharide conjugate vaccine The yield of bound polysaccharide is low.

technical problem

[0007] The technical problem to be solved by the present invention is to provide a method for preparing a group A meningococcal capsular polysaccharide conjugate vaccine, which overcomes the problem of low yield of the combined polysaccharide of the existing group A meningococcal capsular polysaccharide conjugate vaccine. Problem solution

Technical solution

[0008] The technical solution adopted by the present invention to solve the above problems is:

[0009] A method for preparing a group A meningococcal capsular polysaccharide conjugate vaccine, comprising the steps of:

[0010] B1: A group of cerebral capsular polysaccharide is diluted with water for injection to 8 mg/ml

Solution, constant temperature to 20 ° C, adjust the pH to 10.50;

[0011] B2: adding a mass of cyanogen bromide as described in B1 for activation treatment, maintaining ρΗΙΟ.5 activation for 20 minutes, adjusting the pH to 8.5;

[0012] B3: adding an equal volume of equal concentration of azelaic acid dihydrazide solution to the polysaccharide solution described in B1 for coupling reaction, maintaining pH 8.5 for 15 minutes;

[0013] B4: The reactant obtained by B3 is diluted several times - concentrated by ultrafiltration, and the residual cyanogen bromide is removed to obtain a group A cerebral polysaccharide-sebacic acid dihydrazide derivative;

[0014] B5: a B group of meningococcal polysaccharide-sebacic acid dihydrazide derivative obtained in B4 is added with a mass of tetanus toxoid protein solution such as the polysaccharide described by B1, and then diluted with water for injection to make the polysaccharide end The concentration is 3mg/ml;

[0015] B6: The solution obtained by B5 is mixed and placed in an ice bath, and the carbodiimide powder is added to a final concentration of 30 mmol / L, maintained at pH 5.5, and reacted at 2 to 8 ° C for 60 minutes to adjust the pH to 7.0 set 2~ 8 °C for 10 hours;

[0016] B7: The reactant obtained by B6 was separated and purified through a _SepharOSe 4FF gel column, and V was collected. The collected eluate was sterilized by filtration in the vicinity of the eluate to obtain a conjugate of the group A capsular polysaccharide and the carrier protein.

[0017] In the process of binding capsular polysaccharide to tetanus toxoid protein, the bridging agent is very important, and the steric hindrance of the capsular polysaccharide and the tetanus toxoid protein can be overcome by bridging, making the polysaccharide easier to Tetanus toxoid proteins are linked together by covalent bonds. The bridging agent used in the existing conjugate vaccine is adipic hydrazide, and the adipic hydrazide has a hydrazide group at both ends thereof, which easily forms an amide bond with the activated polysaccharide (or tetanus toxoid), and is covalently linked. Together, at present, in the process of preparing a group of ECM vaccines, The efficiency of attaching adipic hydrazide to capsular polysaccharide is very low, and it needs to be added in excess, and the capsular polysaccharide-adipyl hydrazide derivative obtained is not highly efficient in binding to tetanus toxoid protein, resulting in certain resources. Waste, resulting in a low yield of the bound polysaccharide of the group A meningococcal capsular polysaccharide conjugate vaccine, the present invention replaces the conventionally used coupling agent adipic hydrazide with azelaic acid dihydrazide, and the azelaic acid dihydrazide is a A small molecule compound similar in structure to adipic hydrazide, the difference being that there are two more methylene groups on the carbon chain, and the molecular chain is longer, which is used as a conjugate vaccine linker, and can overcome the steric hindrance of biomacromolecules. Increase the binding yield.

Advantageous effects of the invention

Beneficial effect

[0018] In summary, the beneficial effects of the present invention are:

[0019] The present invention uses azelaic acid dihydrazide as a coupling agent in the preparation process of the group A meningococcal conjugate vaccine, the azelaic acid dihydrazide has two methylene groups on the carbon chain compared to the adipic acid hydrazide. The molecular chain is longer, which can overcome the steric hindrance between biological macromolecules and increase the binding yield.

Embodiments of the invention

The invention will be further described in detail below with reference to the embodiments, but the embodiments of the invention are not limited thereto.

[0021] Embodiment:

[0022] A method for preparing a group A meningococcal capsular polysaccharide conjugate vaccine, comprising the steps of:

[0023] B1: A group of cerebral capsular polysaccharide is diluted with water for injection to 8 mg/ml

Solution, constant temperature to 20 ° C, adjust the pH to 10.50;

[0024] B2: adding a mass of cyanogen bromide as described in B1 for activation treatment, maintaining ρΗΙΟ.5 activation for 20 minutes, adjusting the pH to 8.5;

[0025] B3: adding a volumetric, equal concentration of azelaic acid dihydrazide solution of the polysaccharide solution described in B1 for coupling reaction, maintaining pH 8.5 for 15 minutes;

[0026] B4: The reactant obtained by B3 is diluted several times - concentrated by ultrafiltration, and the residual cyanogen bromide is removed to obtain a group A cerebral polysaccharide-sebacic acid dihydrazide derivative;

[0027] B5: a B group of meningococcal polysaccharide-sebacic acid dihydrazide derivative obtained in B4 is added with a mass of tetanus toxoid protein solution such as the polysaccharide described by B1, and then diluted with water for injection to make the polysaccharide end The concentration is 3mg/ml; [0028] B6: The solution obtained by B5 is mixed and placed in an ice bath, and the carbodiimide powder is added to a final concentration of 30 mmol /

L, maintain pH 5.5, react at 8 ° C for 60 minutes, adjust the pH to 7.0 and set at 8 ° C for 10 hours;

[0029] B7: The reactant obtained in B6 was separated and purified through a _SepharOSe 4FF gel column, and V was collected. The collected eluate was sterilized by filtration in the vicinity of the eluate to obtain a conjugate of the group A capsular polysaccharide and the carrier protein.

[0030] Sampling the obtained conjugate of group A capsular polysaccharide and carrier protein to detect polysaccharide content according to pharmacopoeia method

, protein content, free polysaccharide, relative molecular mass, stored at 8 ° C, a total of 3 batches:

[0031] wherein the first batch is added with a polysaccharide amount of 401 mg, in the group A capsular polysaccharide conjugate solution, the conjugate content of the polysaccharide, the conjugate protein content, the conjugate polysaccharide protein ratio, the conjugate free polysaccharide content, the conjugate The relative molecular mass (KD < 0.2 eluent recovery), the yield of the polysaccharide yield are: 126

• 2mg, 221.4mg, 0.57, 4.3% ^ 98.4% ^ 31.5%;

[0032] wherein the second batch is added with a polysaccharide amount of 403 mg, in the group A capsular polysaccharide conjugate solution, the conjugate content of the polysaccharide, the conjugate protein content, the conjugate polysaccharide protein ratio, the conjugate free polysaccharide content, the conjugate The relative molecular mass (KD < 0.2 eluent recovery) and the yield of the polysaccharide yield were: 131

• lmg, 226.0mg, 0.58, 3.7%, 97.6%, 32.5%;

[0033] wherein the third batch is added with a polysaccharide amount of 398 mg, the polysaccharide content of the conjugate in the group A cerebral capsular polysaccharide conjugate, the protein content of the conjugate, the ratio of the polysaccharide protein of the conjugate, the free polysaccharide content of the conjugate, the conjugate The relative molecular mass (KD < 0.2 eluent recovery) and the yield of the polysaccharide yield were: 130

• 3mg, 210.2mg, 0.62, 3.1<3⁄4, 97.2%. 32.7<3⁄4.

[0034] In the same way, using adipic dihydrazide as a coupling agent, a binding reaction is carried out to prepare a conjugate of the conjugate vaccine stock solution, ie, a group of cerebral capsular polysaccharide and a carrier protein, each batch of indicators For:

[0035] The first batch is added with a polysaccharide amount of 400 mg, and the polysaccharide content of the conjugate, the protein content of the conjugate, the ratio of the polysaccharide protein of the conjugate, the free polysaccharide content of the conjugate, and the relative conjugate of the conjugate in the group A capsular polysaccharide conjugate solution The molecular mass (KD<0.2 eluent recovery) and the polysaccharide yield of the conjugate were as follows: 66.1 mg

, 97.2 mg, 0.68, 7.6% ^ 93.8 < 3⁄4, 16.5%;

[0036] The second batch is added with a polysaccharide amount of 402 mg, and the polysaccharide content of the conjugate, the protein content of the conjugate, the ratio of the polysaccharide protein of the conjugate, the free polysaccharide content of the conjugate, and the relative conjugate of the conjugate in the group A capsular polysaccharide conjugate solution The molecular mass (KD<0.2 eluent recovery) and the yield of the polysaccharide yield were: 71.4 mg

, 99.2mg, 0.72, Ί%, 93.2% ^ 17.8%; [0037] The third batch is added with a polysaccharide amount of 399 mg, and the polysaccharide content, the protein content of the binder, the ratio of the polysaccharide protein of the binder, the free polysaccharide content of the binder, and the relative content of the conjugate in the group A capsular polysaccharide conjugate solution The molecular mass (KD < 0.2 eluent recovery) and the yield of the polysaccharide yield were: 70.7 mg, 95.5 mg, 0.74, 6.3% ^ 92.9%, 17.7 < 3⁄4.

[0038] The polysaccharide yield of the group A cerebral capsular polysaccharide conjugate of the invention is more than 30%, compared with the yield of the traditional combination method of 15 to 20%, the polysaccharide yield is greatly improved, and the production is saved. The raw materials make the manpower, raw materials and energy costs of the single-agent finished vaccine greatly reduced.

[0039] As described above, the present invention can be preferably implemented.

Claims

Claim

[Claim 1] A method for preparing a group A meningococcal capsular polysaccharide conjugate vaccine, comprising the steps of:

B1: The group A capsular polysaccharide is diluted with water for injection into a solution of 8 mg/ml, and the temperature is adjusted to 20 ° C, and the pH is adjusted to 10.50;

B2: adding a mass of cyanogen bromide as described in B1 for activation treatment, maintaining ρΗΙΟ.5 activation for 20 minutes, adjusting the pH to 8.5;

B3: adding an equal volume of an equal concentration of azelaic acid dihydrazide solution to the polysaccharide solution described in B1, and performing a coupling reaction at pH 8.5 for 15 minutes;

B4: The reactant obtained by B3 is diluted several times - concentrated by ultrafiltration, and the residual cyanogen bromide is removed to obtain a group A meningococcal polysaccharide-sebacic acid dihydrazide derivative;

B5: Add a solution of a mass of tetanus toxoid protein, such as the polysaccharide described by B1, to the group A meningococcal polysaccharide-sebacic acid dihydrazide derivative obtained in B4, and then dilute with water for injection to make the final concentration of the polysaccharide 3 mg. /ml;

B6: The solution obtained by B5 is mixed and placed in an ice bath, and the carbodiimide powder is added to a final concentration of 3 O mmol / L, maintained at pH 5.5, reacted at 2 to 8 ° C for 60 minutes, and adjusted to pH 7.0. 2 to 8 ° C for 10 hours;

B7: The reactant obtained in B6 was separated and purified on a Sephar _OSe 4FF gel column, and V was collected. The collected eluate was sterilized by filtration in the vicinity of the eluate to obtain a conjugate of the group A capsular polysaccharide and the carrier protein.