CN110917344B

CN110917344B - Liquid vaccine composition and application thereof

Info

Publication number: CN110917344B
Application number: CN201911370164.2A
Authority: CN
Inventors: 范鑫; 韩振; 隋晓斌; 徐树利; 王治伟; 杨帆; 高强; 尹卫东
Original assignee: Sinovac Research & Development Co ltd
Current assignee: Sinovac Research & Development Co ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2022-11-04
Anticipated expiration: 2039-12-26
Also published as: CN110917344A

Abstract

The present invention relates to a liquid vaccine composition comprising: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus, type b haemophilus influenzae capsular polysaccharide-protein conjugate, meningococcal capsular polysaccharide-protein conjugate; the inactivated poliovirus includes inactivated poliovirus Sabin strains type I, type II and type III. The liquid vaccine composition provided by the invention avoids the safety risk and the limitation of capacity caused by the freeze-drying process, and simultaneously avoids the complicated re-dissolving process of the freeze-dried vaccine before immunization. The liquid vaccine composition provided by the invention can prevent six infectious diseases simultaneously, and greatly reduces the workload of inoculation and the burden of children.

Description

Liquid vaccine composition and application thereof

Technical Field

The invention relates to the field of vaccines, and particularly relates to a liquid vaccine composition capable of preventing pertussis, diphtheria, tetanus, poliomyelitis, haemophilus influenzae type b and neisseria meningitidis simultaneously.

Background

Timely vaccination is an effective preventative measure. Pertussis, diphtheria and tetanus combination vaccines (DTPs) have played an important role in the prevention and control of these three infectious diseases as vaccines that were first incorporated into the WHO expanded immunization program (EPI). In the 70 s of the 20 th century, due to serious adverse reactions after whole cell pertussis vaccine (DTwP) inoculation, the phenomenon of resisting vaccination and non-use of vaccine appears in Japan, the Netherlands and other countries, the inoculation rate is reduced, and the disease rate of pertussis is rebounded. At present, most developed countries are prepared into cell-free diphtheria-pertussis-tetanus vaccine (DTaP) with small side reaction by co-purification or chromatography purification, and the cell-free diphtheria-pertussis-tetanus vaccine is used as a basic combined vaccine and a conventional vaccine for immunization. Acellular pertussis vaccines can be divided into co-purified vaccines and chromatographically purified vaccines from the production process point of view. At present, some enterprises in China and Japan adopt a co-purification process, namely, after bacterial culture, protective antigens such as PT, FHA, PRN and the like are precipitated by salting out, then impurities are removed by sucrose density gradient centrifugation, and meanwhile, effective components rich in PT, FHA and PRN are collected. The chromatographic purification is to purify different protective antigens respectively by adopting column chromatography, and then quantitatively proportioning each antigen to prepare the vaccine. The separation and purification method has higher cost, but has the advantages of definite components, easy quality control and less side effect. At present, most developed countries adopt column chromatography to separate and purify each component to produce acellular pertussis vaccines.

Poliomyelitis (WPV) is a widespread and extremely harmful acute infectious disease caused by polioviruses type I, II and III, which can affect people of any age group, but mainly children under 3 years old (accounting for 50% of all cases) and seriously cause irreversible paralysis of limbs and even death. Before the vaccine came into existence, the disease was prevalent worldwide. In the middle and late 50 s, salk and Sabin, two scientists in the United states, developed Inactivated polio Vaccine (IPV for short) and Oral attenuated live polio Vaccine (OPV for short) successively. Since then, people have had powerful weapons to prevent and destroy polio, and practice has proven that both vaccines are effective. The World Health Organization (WHO) initiated a worldwide initiative to eliminate poliomyelitis in 1988, after which activities to eliminate poliomyelitis have made major progress both in reducing the incidence of poliomyelitis and in reducing the spread of poliovirus. In 2007 from 1 month to 2008 from 6 months, 16 countries detected wild strains (WPV) from fecal specimens of AFP cases, only WPVI type and WPVIII type, and no WPVII type was detected worldwide since 1999, and these advances were made thanks to a polio eradication strategy that was commonly practiced throughout the world. In the action of eliminating poliomyelitis (WPV) globally, oral poliomyelitis attenuated live vaccine (OPV) is widely applied, and the incidence rate of the poliomyelitis globally is greatly reduced. However, due to the risk of Vaccine Associated Paralytic Poliomyelitis (VAPP), and Vaccine-derived Poliovirus (VDPV), the use of oral polio attenuated live Vaccine OPV must be stopped in the future to completely eradicate polio. Only by using the inactivated polio vaccine IPV can VAPP and VDPV risks be avoided. The inactivated poliomyelitis vaccine can prevent poliomyelitis IPV outbreak, can generate colony immunity and is the best choice for eliminating subsequent stages of poliomyelitis. At present, manufacturers all over the world produce IPV, but the production virus species used are wild strains. The production of IPV by using wild strains needs to meet the requirement of biological safety level 3 (BSL-3), and the condition of even BSL-4 is not absolutely guaranteed after the poliomyelitis is eliminated all over the world. For the Vaccine manufacturers to be hard to meet the strict biological safety requirements, the currently domestic approved DTaP-IPV/Hib combined Vaccine only has Pentaxim TM produced by SanofiPasteur (SP), in the combined Vaccine, the strain selected by the IPV Vaccine is Salk strain, but the Salk strain belongs to a strong wild strain and has strong toxicity, so that the WHO encourages the Vaccine manufacturers to develop attenuated strain poliomyelitis Inactivated Vaccine Sabin IPV (Inactivated Poliovirus Vaccine, sabin strain, abbreviated as sIPV). To date, most of the strains selected for IPV in the multivalent combination vaccines on the market all over the world are Salk strains. Therefore, the development of a combined vaccine containing the sIPV with higher safety and stability is of great significance.

Haemophilus influenzae (Haemophilus influenzae, hi) is by far the leading causative agent of invasive diseases in humans, most of which are also caused by Haemophilus influenzae type b (Hib). Hib meningitis is the first of bacterial meningitis and has the characteristics of high morbidity, high mortality and high disability rate; it is also characterized by small onset age, high infant infection rate, and the onset age mainly focuses below 5 years, especially below 2 years. Worldwide, an estimated 300 million serious diseases are caused each year, about 38.6 thousands of people die, and the disease becomes a global public health problem. Hib pneumonia plays an important role in childhood infectious diseases in developing countries, accounting for about 20% of all childhood pneumonia, and is an important cause of death in developing countries. In the early 90 s of the last century, hib conjugate vaccines are incorporated into conventional children's immunity programs in countries such as Western Europe and the United states, and the incidence of Hib invasive epidemic diseases is rapidly reduced or disappeared. Currently, the World Health Organization (WHO) is working on driving its inclusion into the planned immunization programs of other countries. In China, the level of Hib natural infection antibodies in children of 3-5 years old is low, and the Hib natural infection antibodies are high risk groups. Clinical research in hospitals in China suggests that Hib meningitis accounts for 51.7% of the children with suppurative meningitis, and 84% of the children are under 2 years old; hib pneumonia accounts for 34.3% in children, so that the necessity of Hib conjugate vaccination is urgently needed to be solved in China.

Epidemic cerebrospinal meningitis is an acute respiratory infectious disease caused by neisseria meningitidis (neisserial meningitidis). For over a hundred years, it has been prevalent or scattered around the world and can cause septicemia and meningitis after infection with pathogenic bacteria. The susceptible population is mainly children, and the outbreak fatality rate is the highest and can reach 40-60%. The incidence of diseases in continents in the world is 1/10-10/10 ten thousand, the total incidence is 5-15%, and up to 20% of meningitis patients have nervous system sequelae including intelligence impairment, deafness and the like. Serological classification according to capsular polysaccharide type can be divided into 13 serotypes, wherein group A, group B and group C account for about 90% of the epidemic flora. China has developed 5 national epidemic encephalitis in 1938, 1949, 1959, 1967, and 1977. The disease rate is the most serious in 1967 spring, the disease rate is as high as 403/10 ten thousands, and the disease death rate is 5.49%. In China, over 90% of the past cases are pathogenic to group A germs, and epidemic encephalitis sometimes occurs to group B germs and group C germs. The incidence of epidemic encephalitis in group C was significantly increased since 2003. Group a and group C currently account for more than 50% of all serogroups, and group C tends to be further elevated. The most effective method for preventing epidemic encephalitis is vaccination at present. Epidemic encephalitis vaccines have been brought into the national immune program in China since 2007, and are generally inoculated to children of the right age nationwide.

Due to the distribution difference of epidemic diseases and the limitation of research difficulty, no combined vaccine (DTaP-sIPV-Hib-Men) for adsorbing acellular diphtheria-poliovirus inactivated vaccine-b-type haemophilus influenzae (combined) -meningococcus polysaccharide (combined) is marketed at home and abroad at present. At present, although there are similar products (DTaP/Hib/Men) in development at home and abroad, the Hib and Men components are still prepared into corresponding combined vaccines by adopting a freeze-drying process; the commercially available freeze-dried vaccines need to be redissolved before immunization, and the redissolution process is complicated; meanwhile, based on the safety risk and the limitation of capacity introduced by a freeze-drying process, the research of adsorbing the acellular pertussis-poliovirus inactivated vaccine-b-type haemophilus influenzae (combined) -meningococcal polysaccharide (combined) liquid combined vaccine is carried out, and the method has good social value and economic value.

Disclosure of Invention

The purpose of the present invention is to provide a liquid vaccine composition that can prevent pertussis, diphtheria, tetanus, poliomyelitis, haemophilus influenzae type b, and Neisseria meningitidis at the same time.

The 6 infectious diseases are still the important causes of infant death, and are brought into the state EPI, 13 injections are needed after inoculation (3 times of basic immunity and 1 times of boosting immunity; poliomyelitis: 3 times of basic immunity and 1 times of boosting immunity; haemophilus influenzae type b: 3 times of basic immunity and 1 times of boosting immunity; epidemic cerebrospinal meningitis of A group and C group: 1 times of immunity). In order to improve the condition that the inoculation times are various and avoid wrong inoculation and missed inoculation, the liquid vaccine composition provided by the invention can simultaneously prevent the liquid vaccine composition of the infectious diseases, and the liquid vaccine composition is inoculated for 4 times (3 times of basic immunization and 1 time of boosting immunization) according to the inoculation procedure. Greatly reducing the workload of inoculation and the burden of children.

Specifically, the invention provides a liquid vaccine composition, which is a liquid and comprises: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus, type b haemophilus influenzae capsular polysaccharide-protein conjugate, meningococcal capsular polysaccharide-protein conjugate; the inactivated poliovirus includes inactivated Sabin strain polioviruses of type I, type II and type III.

The invention provides a liquid vaccine composition, which is liquid, and the antigen of the liquid vaccine composition consists of the following substances: diphtheria toxoid, tetanus toxoid, pertussis toxin, filamentous hemagglutinin, pertactin, inactivated poliovirus, type b haemophilus influenzae capsular polysaccharide-protein conjugate, meningococcal capsular polysaccharide-protein conjugate; the inactivated poliovirus consists of inactivated Sabin strain polioviruses of type I, type II and type III.

According to the invention, the Sabin strain has the advantage of high safety, in order to ensure the immunogenicity of the Sabin strain and simultaneously avoid the interference effect of the Sabin strain and other antigens in the combined vaccine as much as possible, the antigen proportion of the poliovirus of the Sabin strains of type I, type II and type III in the composition is preferably (4-15): (11-45): (11 to 45), more preferably 1: (2.5-3.5): (2.5-3.5). As a particularly preferred embodiment of the present invention, the following components are included in each 0.5mL of the liquid vaccine composition: type I Sabin strain poliovirus 4-15DU, type II Sabin strain poliovirus 11-45DU, and type III Sabin strain poliovirus 11-45 DU.

The haemophilus influenzae type b capsular polysaccharide-protein conjugate is formed by conjugating haemophilus influenzae type b capsular polysaccharide with physiologically acceptable carrier protein. The physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, nontoxic variant of diphtheria toxin CRM197, tetanus toxoid, meningococcal outer membrane protein.

The meningococcus capsular polysaccharide-protein conjugate is formed by conjugating meningococcus capsular polysaccharide and physiologically acceptable carrier protein. The physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, nontoxic variant CRM197 of diphtheria toxin, tetanus toxoid, and meningococcal outer membrane protein.

The meningococcus capsular polysaccharide-protein conjugate can specifically refer to one or more of a group A meningococcus capsular polysaccharide-protein conjugate, a group B meningococcus capsular polysaccharide-protein conjugate, a group C meningococcus capsular polysaccharide-protein conjugate, a group Y meningococcus capsular polysaccharide-protein conjugate and a group W135 meningococcus capsular polysaccharide-protein conjugate. As a specific embodiment of the invention, the meningococcal capsular polysaccharide-protein conjugates include group a meningococcal capsular polysaccharide-protein conjugates and group C meningococcal capsular polysaccharide-protein conjugates.

The vaccine composition provided by the invention can further comprise one or more of aluminum adjuvant, soluble phosphate buffer solution, sodium chloride and stabilizer on the basis of the antigen stock solution. In particular, adjuvants, such as adjuvants comprising aluminium hydroxide and/or aluminium phosphate; practice of the invention shows that when the aluminum content is 0.3-1.25 mg/ml, preferably 0.4-0.8 mg/ml, the titer of each type of antigen can be kept at a higher quality standard while the safety of the liquid vaccine composition is ensured. The vaccine composition may also include a quantity of soluble phosphate buffer to maintain the pH within a reasonable range (e.g., pH 5.8-7.2), and a quantity of sodium chloride or equivalent to maintain osmotic pressure. The stabilizer can be selected from vitamins and/or amino acids, preferably amino acids; the invention discovers that the stability of the polysaccharide-protein conjugate of the haemophilus influenzae type b and the polysaccharide-protein conjugate of the meningococcus in a liquid preparation can be obviously improved by adding the amino acid into the liquid composition, so that the overall stability of the liquid composition is improved. The stabilizer can also be selected from an M199 culture medium; practice shows that when 5% -20% of 10X M199 culture medium concentrated solution is added into the combined vaccine, the stability of the poliovirus antigen is remarkably improved, so that the stability of the whole liquid vaccine composition is significantly improved.

As a preferred embodiment of the present invention, the liquid vaccine composition comprises the following components:

the liquid vaccine composition provided by the invention can be used for basic immunity and boosting immunity.

The invention discovers through a great deal of practice that when the proportion and the dosage of the pertussis toxin, filamentous hemagglutinin and pertactin contained in the composition are optimally adjusted, the vaccine composition can further meet the requirements of basic immunity or enhanced immunity on the basis of ensuring the safety.

When the liquid vaccine composition of the present invention is used for preparing a basic immune vaccine, the mass ratio of pertussis toxin, filamentous hemagglutinin, and pertactin contained in the composition is preferably (20 to 25): (20-25): (3-8). Preferably, the composition contains 20-25 mug of pertussis toxin, 20-25 mug of filamentous hemagglutinin and 3-8 mug of pertactin per 0.5ml single dose. Further preferably, the composition contains 25 μ g of pertussis toxin, 25 μ g of filamentous hemagglutinin and 8 μ g of pertactin per 0.5ml single dose.

As a preferred embodiment of the present invention, the liquid vaccine composition can be used for basic immunization, and the following components are contained in the composition per 0.5ml single dose:

when the liquid vaccine composition is used for preparing a booster vaccine, the mass ratio of pertussis toxin, filamentous hemagglutinin and bordetella pertussis adhesin in the composition is preferably (2.5-8): (5-8): (2.5-3). Preferably, the composition contains 2.5-8 mug of pertussis toxin, 5-8 mug of filamentous hemagglutinin and 2.5-3 mug of pertactin per 0.5ml single dose. Further preferably, the composition contains 8 μ g of pertussis toxin, 8 μ g of filamentous hemagglutinin and 2.5 μ g of pertactin per 0.5ml single dose.

As a preferred embodiment of the present invention, the liquid vaccine composition can be used for boosting immunity, and the composition comprises the following components in a single dose of every 0.5 ml:

the invention also provides a method for preparing the liquid vaccine composition.

The method specifically comprises the following steps: mixing liquid raw materials including pertussis toxin, filamentous hemagglutinin, pertactin, diphtheria toxoid raw liquid, tetanus toxoid raw liquid, inactivated Sabin I strain poliovirus raw liquid, inactivated Sabin II strain poliovirus raw liquid, inactivated Sabin III strain poliovirus raw liquid, b type haemophilus influenzae polysaccharide-protein conjugate raw liquid and meningococcus polysaccharide-protein conjugate raw liquid. The compositions provided by the present invention are liquid formulations, wherein the various raw materials are also prepared in liquid form and then directly mixed.

After the liquid raw materials are mixed, substances such as an adjuvant, a soluble phosphate buffer solution, sodium chloride, a stabilizer and the like are further added, so that a stable liquid vaccine composition suitable for human bodies can be obtained.

The invention protects the application of the liquid vaccine composition in the preparation of basic immunity vaccines.

The invention protects the application of the liquid vaccine composition in preparing a booster vaccine.

The invention provides a vaccine kit comprising a first chamber and a second chamber; the first chamber contains a basic immunity vaccine prepared from the liquid vaccine composition; the second chamber contains a booster vaccine prepared from the liquid vaccine composition.

The invention provides a vaccine kit, comprising a first chamber, a second chamber and a third chamber; the first chamber and the second chamber respectively contain basic immunity vaccines prepared by the liquid vaccine composition; the third chamber contains a booster vaccine prepared from the liquid vaccine composition.

The invention provides a vaccine kit, which comprises a first chamber, a second chamber, a third chamber and a fourth chamber; the first chamber, the second chamber and the third chamber respectively contain basic immunity vaccines prepared by the liquid vaccine composition; the fourth compartment contains a booster vaccine prepared from the liquid vaccine composition.

During actual inoculation, the vaccine kit provided by the invention can efficiently complete the vaccination, and avoid misconception or missed inoculation. For example: the basic immunization is carried out by using the basic immunization vaccine in the first chamber (or the first chamber and the second chamber are used in sequence, or the first chamber, the second chamber and the third chamber are used in sequence) in the kit, and then the boosting immunization is carried out by using the boosting immunization vaccine in the second chamber (or the third chamber or the fourth chamber) in the kit.

Compared with the prior art, the vaccine composition provided by the invention is a liquid preparation, can maintain the stability of the property of the liquid preparation in each link of preparation, transportation, storage and use, avoids the safety risk and the limitation of capacity caused by the introduction of a freeze-drying process, and simultaneously avoids the complicated re-dissolving process of the freeze-dried vaccine before immunization. The liquid vaccine composition provided by the invention can simultaneously prevent six infectious diseases caused by pertussis, diphtheria, tetanus, poliomyelitis, haemophilus influenzae type b and neisseria meningitidis, and greatly reduces the workload of inoculation and the burden of children.

Drawings

FIG. 1 shows the results of measuring the type I D antigen content of sIPV in example 9;

FIG. 2 shows the results of measuring the type II D antigen content of sIPV in example 9;

FIG. 3 shows the measurement results of the type III D antigen content of sIPV in example 9;

figure 4 is the results of the sIPV neutralizing antibody titer assay described in example 11;

figure 5 shows the results of pertussis (aP) antibody titer assays according to the immunization procedure described in example 11; wherein, aP-PT represents pertussis toxin, aP-FHA represents filamentous hemagglutinin, aP-PRN represents pertactin;

FIG. 6 shows the results of the Diphtheria (DT), tetanus (TT) antibody titer assays of the immunization procedure described in example 11;

FIG. 7 shows the results of the antibody titer assays for Hib and Group A (AGroup) and Group C (C Group) Neisseria meningitidis according to the immunization protocol described in example 11.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

This example provides a liquid vaccine formulation comprising the following components in an amount per 1 human dose:

8 mug of pertussis toxin, 8 mug of filamentous hemagglutinin, 2.5 mug of pertactin, 2Lf of diphtheria toxoid, 5Lf of tetanus toxoid, 7.5DU of inactivated poliovirus type I stock solution, 22.5DU of inactivated poliovirus type II stock solution, 22.5DU of inactivated poliovirus type III stock solution, 10 mug of A group meningococcus polysaccharide-protein conjugate, 10 mug of C group meningococcus polysaccharide-protein conjugate, 10 mug of B type haemophilus influenzae polysaccharide-protein conjugate, 0.6mg/ml of aluminum content, a proper amount of sodium chloride aqueous solution, and a pH value adjusted to 5.8-7.2 by phosphate buffer. The vaccine may also contain appropriate amount of stabilizer such as M199 culture medium and amino acids.

The combined vaccine is mixed evenly before use and is used for intramuscular injection.

The liquid vaccine formulations provided in this example can be used to boost immunity.

Example 2

8 mug of pertussis toxin, 8 mug of filamentous hemagglutinin, 2.5 mug of pertactin, 2Lf of diphtheria toxoid, 5Lf of tetanus toxoid, 15DU of inactivated poliovirus type I stock solution, 45DU of inactivated poliovirus type II stock solution, 45DU of inactivated poliovirus type III stock solution, 15 mug of A-group meningococcus polysaccharide-protein conjugate, 15 mug of C-group meningococcus polysaccharide-protein conjugate, 15 mug of b-type haemophilus influenzae polysaccharide-protein conjugate, 0.8mg/ml of aluminum content, a proper amount of sodium chloride aqueous solution, and a pH value adjusted to 5.8-7.2 by phosphate buffer. The vaccine may also contain appropriate amount of stabilizer such as M199 culture medium and amino acids.

Example 3

25 mug of pertussis toxin, 25 mug of filamentous hemagglutinin, 8 mug of pertactin, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 15DU of inactivated poliovirus type I stock solution, 45DU of inactivated poliovirus type II stock solution, 45DU of inactivated poliovirus type III stock solution, 10 mug of A group meningococcus polysaccharide-protein conjugate, 10 mug of C group meningococcus polysaccharide-protein conjugate, 10 mug of b type haemophilus influenzae polysaccharide-protein conjugate, 0.8mg/ml of aluminum content, a proper amount of sodium chloride aqueous solution, and a phosphate buffer for adjusting the pH value to 5.8-7.2. The vaccine may also contain appropriate amount of stabilizer such as M199 culture medium and amino acids.

The liquid vaccine formulations provided in this example can be used for basic immunization.

Example 4

25 mug of pertussis toxin, 25 mug of filamentous hemagglutinin, 8 mug of pertactin, 12.5Lf of diphtheria toxoid, 3.5Lf of tetanus toxoid, 7.5DU of inactivated poliovirus type I stock solution, 22.5DU of inactivated poliovirus type II stock solution, 22.5DU of inactivated poliovirus type III stock solution, 15 mug of A-group meningococcus polysaccharide-protein conjugate, 15 mug of C-group meningococcus polysaccharide-protein conjugate, 15 mug of b-type haemophilus influenzae polysaccharide-protein conjugate, 0.8mg/ml of aluminum content, a proper amount of sodium chloride aqueous solution and a pH value adjusted to 5.8-7.2 by phosphate buffer. The vaccine may also contain appropriate amount of stabilizer such as M199 culture medium and amino acids.

Example 5

This example provides a method of preparing each of the monovalent vaccine stock solutions referred to in examples 1 to 4.

1. Diphtheria toxoid stock solution

Opening diphtheria bacillus, transferring 1-3 generations of diphtheria bacillus in a seed tube of a toxin-producing culture medium, transferring the diphtheria bacillus to the toxin-producing culture medium to prepare seeds for production, culturing the seeds in a culture tank by using liquid, culturing the seeds in a maltose comprehensive culture medium at 34-36 ℃ for 45-52 hours, adding formaldehyde to sterilize after the culture is finished, adding ammonium sulfate to precipitate impure protein into a culture, centrifugally collecting supernatant, performing ultrafiltration concentration, adding ammonium sulfate to salt out and precipitate diphtheria toxin protein, centrifugally collecting precipitate, adding PB buffer solution to dissolve the precipitate, performing ultrafiltration to remove ammonium sulfate in toxin, adding formaldehyde, standing for 35-37 ℃, detoxifying for 30 days, removing formaldehyde in toxoid by ultrafiltration, and performing sterilization filtration to obtain diphtheria toxoid raw liquid.

2. Tetanus toxoid stock solution

Starting clostridium tetani, transferring 1-3 generations of clostridium tetani in a seed tube of a toxin-producing culture medium, transferring to the toxin-producing culture medium to prepare seeds for production, culturing the seeds by adopting a culture tank liquid, culturing the seeds for 62-72 hours at 34-36 ℃ by adopting a double Chen Peiyang medium, adding formaldehyde for sterilization after the culture is finished, filtering the culture liquid to remove a sterilization body, adding ammonium sulfate to precipitate toxin protein, centrifugally collecting precipitate, adding formaldehyde after removing the ammonium sulfate in the toxin by ultrafiltration, standing for 35-37 ℃, detoxifying for 30 days, removing the formaldehyde in the toxoid by ultrafiltration, and sterilizing and filtering to obtain a tetanus toxoid stock solution.

3. Acellular pertussis vaccine stock solution

1) The method comprises the steps of starting Bordetella pertussis, inoculating the Bordetella pertussis to an improved bag-ginger culture medium or an active carbon semi-comprehensive culture medium, culturing at 35-37 ℃ for no more than 48 hours, inoculating the Bordetella pertussis to a CPB culture medium for two generations, culturing for 18-26 hours each generation, performing amplification culture to prepare enough production seeds, inoculating the seeds to a fermentation tank for liquid culture, culturing for two days at 37 ℃ by adopting the CPB culture medium, adding thimerosal or formaldehyde into a culture for sterilization treatment after the culture is finished, centrifuging the obtained culture, and storing the thalli and supernatant obtained by centrifugation at 2-8 ℃ for further purification.

2) Purification of pertussis toxin and filamentous hemagglutinin: ultrafiltering and concentrating the supernatant, then loading the supernatant into a Capto SP chromatographic column, eluting a mobile phase by using a urea-containing phosphate buffer solution in a sodium chloride gradient manner, respectively collecting elution components containing pertussis toxin and filamentous hemagglutinin, loading the elution components containing pertussis toxin into a Capto MMC chromatographic column, eluting the buffer solution by using a Tris-HCL buffer solution in a sodium chloride gradient manner, and collecting a pertussis toxin elution peak; and (4) loading the elution peak containing the filamentous hemagglutinin to a hydroxyapatite chromatography column for purification, and collecting the elution peak.

3) Separation and purification of pertussis adhesin: releasing the thalli with heat at 60 ℃, ultrafiltering and concentrating, loading onto a Capto adhere chromatographic column, collecting an elution component containing the pertussis adhesin by taking an acetic acid buffer solution as a mobile phase, loading onto a Capto SP chromatographic column, taking the acetic acid buffer solution as the mobile phase, performing gradient elution by using sodium chloride, and collecting an elution peak, namely the separated and purified pertussis adhesin.

4) Preparing acellular pertussis stock solution: detoxication of pertussis toxin by glutaraldehyde, treatment of filamentous hemagglutinin and pertussis adhesin by formaldehyde, degerming and filtering to obtain acellular pertussis stock solution.

4. Poliovirus stock solution

Poliovirus type I stock solution:

1) Cell preparation: taking 1 or more cell tubes in a working cell bank, recovering the cell tubes, then culturing the cell tubes in a first-stage cell reactor at 36.5 +/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml; digesting the cells in the first-stage cell reactor by trypsin, inoculating the cells to the second-stage cell reactor, and culturing at 36.5 +/-0.5 ℃ until the cell concentration is 2-10 multiplied by 106 cells/ml; digesting the cells in the second-stage cell reactor by trypsin, inoculating the cells to the third-stage cell reactor, culturing at 36.5 +/-0.5 ℃ until the cell concentration is 1-5 multiplied by 106 cells/ml, and inoculating the virus.

2) Virus culture and harvest: replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, inoculating Vero cells to the I-type poliovirus working seed batch virus seeds according to MOI = 0.002-0.02, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

3) Clarifying and purifying a virus harvest solution: the harvest liquid is concentrated by 50-200 times by using a 300-500kDa ultrafiltration membrane package after being centrifuged and clarified. And (3) after the concentration is finished, purifying and separating the virus by adopting sucrose density gradient centrifugation, wherein the sucrose gradient is specifically that the first gradient of sucrose density gradient liquid is 30% of sucrose, the second gradient is 55% of sucrose, the centrifugation is carried out for 10 hours at 30000rpm, and a zone where the virus is located is collected. And (3) carrying out anion exchange chromatography on the virus liquid collected by density gradient centrifugation, monitoring the wavelengths of 280nm and 254nm, and collecting flow-through liquid to obtain virus chromatography liquid. And concentrating the virus chromatography liquid to obtain a purified liquid. The anion exchange chromatography may be DEAE Sepharose Fast Flow, Q Sepharose Fast Flow or other anion exchange chromatography. In one embodiment of the invention, the anion exchange chromatography used is DEAE Sepharose Fast Flow.

4) Virus inactivation: sterilizing and filtering the purified solution with the particle size of 0.22 mu m, adding a formaldehyde solution to ensure that the final concentration of free formaldehyde is 90 mu g/ml, inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃, and continuously inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃ after sterilizing and filtering to obtain the monovalent stock solution.

Poliovirus type II stock solution:

2) Virus culture and harvest: and replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, inoculating Vero cells into II type poliovirus working seed batch virus seeds according to MOI = 0.01-0.1, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

3) Clarifying and purifying a virus harvest solution: the harvest liquid is concentrated by 50-200 times by using a 300-500kDa ultrafiltration membrane package after being centrifuged and clarified. And after the concentration is finished, performing centrifugal purification and separation on the virus by adopting a sucrose density gradient, wherein the sucrose gradient is specifically a sucrose density gradient solution with a first gradient of 30% sucrose and a second gradient of 55% sucrose, centrifuging at 30000rpm for 10 hours, and collecting a zone where the virus is located. And (3) carrying out anion exchange chromatography on the virus liquid collected by density gradient centrifugation, monitoring the wavelengths of 280nm and 254nm, and collecting flow-through liquid to obtain virus chromatography liquid. And concentrating the virus chromatography liquid to obtain a purified liquid. The anion exchange chromatography may be DEAE Sepharose Fast Flow, Q Sepharose Fast Flow or other anion exchange chromatography. In one embodiment of the invention, the anion exchange chromatography used is DEAE Sepharose Fast Flow.

Poliovirus type III stock solution:

2) Virus culture and harvest: and replacing the cell culture solution in the third-stage cell reactor with a maintenance solution, taking III-type poliovirus working seed batch virus seeds MOI = 0.05-0.5, inoculating Vero cells, and culturing at 32.5 +/-0.5 ℃. Culturing the virus for 2-4 days, and harvesting cell supernatant, namely the monovalent harvesting solution of the poliovirus Sabin strain.

5) Virus inactivation: sterilizing and filtering the purified solution with the particle size of 0.22 mu m, adding a formaldehyde solution to ensure that the final concentration of free formaldehyde is 90 mu g/ml, inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃, and continuously inactivating for 6 days at the temperature of 37.0 +/-1.0 ℃ after sterilizing and filtering to obtain the monovalent stock solution.

5. Meningococcal polysaccharide-protein conjugate vaccine stock solution

Meningococcal group a conjugate vaccine stock solution:

1) Starting a batch strain of A group meningococcus working seeds, inoculating the batch strain to a epidemic cerebrospinal meningitis semi-comprehensive culture medium, culturing at the temperature of 35-37 ℃ for 16-24 hours, then performing third-generation amplification culture to prepare seeds for production, inoculating the seeds to a fermentation tank for culture at the temperature of 35-37 ℃, and adding formaldehyde for sterilization after the culture time is 6-12 hours; centrifuging a sterilized culture solution to collect supernatant, adding cetyl trimethyl ammonium bromide to the final concentration of 1.0g/L, fully stirring, standing overnight, centrifuging to collect polysaccharide, stirring the precipitated polysaccharide with a calcium chloride solution for 3 hours to dissociate polysaccharide and CTAB, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 25%, standing overnight at 2-8 ℃, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 75-80%, fully shaking to precipitate polysaccharide, standing for more than 18 hours, centrifuging to collect precipitate, then washing with absolute ethanol and acetone for three times respectively, and drying to obtain crude polysaccharide; dissolving crude sugar in 10% saturated central sodium acetate solution, adding cold phenol solution according to the proportion of 1:1 (v/v), shaking, uniformly mixing, centrifuging, collecting supernatant, extracting for 1-3 times, clarifying the supernatant, collecting the supernatant, removing residual phenol and nucleic acid by ultrafiltration membrane ultrafiltration, adding 4mol/L sodium chloride solution into the polysaccharide solution after ultrafiltration until the final concentration of sodium chloride is 0.3mol/L, then adding 95% ethanol until the final concentration of ethanol is 75% (v/v), fully mixing, standing overnight at 2-8 ℃, precipitating refined sugar, centrifuging, collecting precipitate, then sequentially washing twice by absolute ethyl alcohol and acetone, and drying to obtain the refined sugar.

2) Dissolving refined sugar in water for injection, and mixing the refined sugar with the polysaccharide: cyanogen bromide =1, 0.5 (w/w), adding hydrogen bromide solution, maintaining the temperature at 23 ± 3 ℃ and the pH at 10.8 ± 0.2, reacting for 30 minutes, and activating, wherein after the activation is completed, the ratio of polysaccharide: adding 2.8% oxalyl hydrazine solution into adipoyl hydrazine =1 (w/w) in a ratio of 3.5, maintaining the temperature at 23 +/-3 ℃ and the pH at 8.5 +/-0.2, reacting for 15 minutes, performing derivatization, clarifying and filtering, using 0.05mol/L sodium chloride solution as ultrafiltrate, and performing ultrafiltration by using an ultrafiltration membrane package to remove cyanogen bromide, namely obtaining a polysaccharide derivative according to the following formula: protein =1 ratio between 0.8 and 1.2 (w/w) CRM197 protein dosage was calculated and added to the polysaccharide derivative solution, as polysaccharide: carbodiimide is added according to the proportion of carbodiimide =1 (w/w) 10, the reaction temperature is maintained at 5 +/-3 ℃ and the pH value is maintained at 5.7 +/-0.2, the reaction is carried out for 60 minutes, then the pH value is adjusted to 6.9 +/-0.1, the reaction is stopped, after clarification and filtration, the carbodiimide is removed by ultrafiltration by 0.15mol/L sodium chloride solution, then the polysaccharide protein conjugate solution is purified by a Sepharose 4FF gel chromatography column, 0.15mol/L sodium chloride solution is used as a mobile phase, the absorption value of 280nm is detected, the elution peak near V0 is collected, the eluents obtained by each chromatography are combined to obtain a purified conjugate, and after degerming and filtration, the vaccine stock solution of the meningococcus group A conjugate is obtained.

Meningococcal group C conjugate vaccine stock solution:

1) Starting a batch strain of working seeds of the C group meningococcus, inoculating the batch strain to a epidemic cerebrospinal meningitis semi-comprehensive culture medium, culturing at the temperature of 35-37 ℃ for 16-24 hours, then performing third-generation amplification culture to prepare seeds for production, inoculating the seeds to a fermentation tank for culture at the temperature of 35-37 ℃, and adding formaldehyde for sterilization after the culture time is 6-12 hours; centrifuging a sterilized culture solution to collect supernatant, adding cetyl trimethyl ammonium bromide to the final concentration of 1.0g/L, fully stirring, standing overnight, centrifuging to collect polysaccharide, stirring the precipitated polysaccharide with a calcium chloride solution for 3 hours to dissociate polysaccharide and CTAB, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 25%, standing overnight at 2-8 ℃, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 75-80%, fully shaking to precipitate polysaccharide, standing for more than 18 hours, centrifuging to collect precipitate, then washing with absolute ethanol and acetone for three times respectively, and drying to obtain crude polysaccharide; dissolving crude sugar in 10% saturated central sodium acetate solution, adding cold phenol solution according to the proportion of 1:1 (v/v), shaking, uniformly mixing, centrifuging, collecting supernatant, extracting for 1-3 times, clarifying the supernatant, collecting the supernatant, removing residual phenol and nucleic acid by ultrafiltration membrane ultrafiltration, adding 4mol/L sodium chloride solution into the polysaccharide solution after ultrafiltration until the final concentration of sodium chloride is 0.3mol/L, then adding 95% ethanol until the final concentration of ethanol is 75% (v/v), fully mixing, standing overnight at 2-8 ℃, precipitating refined sugar, centrifuging, collecting precipitate, then sequentially washing twice by absolute ethyl alcohol and acetone, and drying to obtain the refined sugar.

2) Dissolving refined sugar in water for injection, and mixing the following raw materials according to the weight ratio of polysaccharide: cyanogen bromide =1 (w/w), adding cyanogen bromide solution, maintaining the temperature at 23 + -3 deg.C and pH at 10.8 + -0.2, reacting for 30 minutes, activating, and adding polysaccharide: adding 2.8% oxalyl hydrazine solution into adipoyl hydrazine =1 (w/w) in a ratio of 3.5, maintaining the temperature at 23 +/-3 ℃ and the pH at 8.5 +/-0.2, reacting for 15 minutes, performing derivatization, clarifying and filtering, using 0.05mol/L sodium chloride solution as ultrafiltrate, and performing ultrafiltration by using an ultrafiltration membrane package to remove cyanogen bromide, namely obtaining a polysaccharide derivative according to the following formula: protein =1 ratio between 0.8 and 1.2 (w/w) CRM197 protein dosage was calculated and added to the polysaccharide derivative solution, as polysaccharide: carbodiimide is added according to the proportion of carbodiimide =1 (w/w) 10, the reaction temperature is maintained at 5 +/-3 ℃ and the pH value is maintained at 5.7 +/-0.2, the reaction is carried out for 60 minutes, then the pH value is adjusted to 6.9 +/-0.1, the reaction is stopped, after clarification and filtration, the carbodiimide is removed by ultrafiltration by 0.15mol/L sodium chloride solution, then the polysaccharide protein conjugate solution is purified by a Sepharose 4FF gel chromatography column, 0.15mol/L sodium chloride solution is used as a mobile phase, the absorption value of OD280nm is detected, elution peaks near V0 are collected, eluents obtained by each chromatography are combined to obtain a purified conjugate, and after degerming and filtration, the vaccine stock solution of the C-group meningococcus conjugate is obtained.

6. Haemophilus influenzae type b polysaccharide-protein conjugate vaccine stock solution

1) Starting a working seed batch strain of the haemophilus influenzae type b, inoculating the working seed batch strain to a Hib comprehensive culture medium, culturing for 16-24 hours at the temperature of 35-37 ℃ in a 5-8% carbon dioxide environment, then performing third-generation amplification culture to prepare seeds for production, inoculating the seeds to a fermentation tank for culture at the temperature of 35-37 ℃ for 6-12 hours, and adding formaldehyde for sterilization; centrifuging sterilized culture solution to collect supernatant, then adding cetyl trimethyl ammonium bromide to the final concentration of 1.0g/L, fully stirring, standing overnight, centrifuging to collect polysaccharide, stirring the precipitated polysaccharide with sodium chloride solution for 3 hours to dissociate polysaccharide and cetyl trimethyl ammonium bromide, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 25% (v/v), standing overnight at 2-8 ℃, centrifuging to collect supernatant, adding ethanol to the supernatant to the final concentration of 75-80% (v/v), fully shaking to precipitate polysaccharide, standing overnight, centrifuging to collect precipitate, washing with absolute ethanol and acetone for three times respectively, and drying to obtain crude polysaccharide; dissolving crude sugar in 10% saturated central sodium acetate solution, adding cold phenol solution according to the proportion of 1:1 (v/v), oscillating, mixing uniformly, centrifuging, collecting supernatant, extracting for 1-3 times, clarifying the supernatant, collecting the supernatant, removing residual phenol and nucleic acid by ultrafiltration membrane filtration, adding 4mol/L sodium chloride solution into the polysaccharide solution after ultrafiltration until the final concentration of sodium chloride is 0.3mol/L, then adding 95% ethanol until the final concentration of ethanol is 75% (v/v), fully mixing uniformly, standing overnight at 2-8 ℃, precipitating refined sugar, centrifuging, collecting precipitate, then sequentially adding absolute ethanol and acetone, washing twice, and drying to obtain the refined sugar.

2) Dissolving refined sugar in water for injection, and mixing the obtained solution with polysaccharide: cyanogen bromide =1 (w/w), adding cyanogen bromide solution, maintaining the temperature at 23 + -3 deg.C and pH at 10.8 + -0.2, reacting for 30 minutes, activating, and adding polysaccharide: adding 2.8% oxalyl hydrazine solution into oxalyl hydrazine =1 (w/w) in a ratio of 3.5, maintaining the temperature at 23 +/-3 ℃ and the pH at 8.5 +/-0.2 for 15 minutes to perform derivatization, and performing ultrafiltration by using 0.05mol/L sodium chloride to remove cyanogen bromide after clarification and filtration to obtain a polysaccharide derivative; according to the polysaccharide: protein =1, 0.8-1.2 (w/w), calculated for tetanus toxoid, added to the polysaccharide derivative solution, as the ratio of polysaccharide: carbodiimide is added according to the proportion of carbodiimide =1 (w/w) 10, the reaction temperature is maintained at 5 +/-3 ℃, the pH value is maintained at 5.7 +/-0.2, the reaction is carried out for 60 minutes, then the pH value is adjusted to 6.9 +/-0.1, the reaction is stopped, then the polysaccharide-protein conjugate is purified by a Sepharose 4FF gel chromatography column, 0.15mol/L sodium chloride solution is used as a mobile phase, the absorption value of OD280nm is monitored, elution peaks near V0 are collected, eluents obtained by each chromatography are combined to obtain a purified conjugate, and the purified conjugate is sterilized and filtered to obtain the Haemophilus influenzae type b conjugate vaccine stock solution.

Example 6

This example provides a method of preparing the liquid vaccine compositions of examples 1-4, comprising the steps of:

1) Mixing pertussis toxin, filamentous hemagglutinin, pertussis adhesin, diphtheria toxoid stock solution, tetanus toxoid stock solution, inactivated poliovirus type I stock solution, inactivated poliovirus type II stock solution, inactivated poliovirus type III stock solution, meningococcal a conjugate vaccine stock solution, meningococcal C conjugate vaccine stock solution and haemophilus influenzae type b conjugate vaccine stock solution, adding an aluminum salt adjuvant diluted by a sodium chloride aqueous solution, adding a phosphate buffer solution and a stabilizer, and adjusting the pH to 5.8-7.2 to obtain a semi-finished product;

2) And (3) subpackaging the semi-finished product with a penicillin bottle or a prefilled syringe.

Example 7

This example analyzes the effect of the antigen content of different Sabin strains on the IPV immunological efficacy of the combination vaccine.

In this example, DTaP-sIPV-Hib-AC combination vaccine samples (samples 1, 2, and 3) containing different type I, II, and III D antigens were prepared, and the antigen content of the three combination vaccines is shown in table 1, using a commercially available IPV vaccine as a control. Wistar rats are immunized with 10 test products and control products respectively, each Wistar rat is injected with 0.5ml, blood is collected 21 days after immunization, serum is separated, cell-mediated poliomyelitis neutralizing antibody titer in rats is detected, and ED50 value is calculated.

Table 1: antigen content of three combination vaccines

The detection results of the titer of the neutralizing antibody of the poliomyelitis types I, II and III are shown in tables 2, 3 and 4, and the results show that the neutralizing antibodies of the poliomyelitis types I, II and III in rats immunized by the three DTaP-sIPV-Hib-AC combined vaccines are not lower than those of the control commercial IPV vaccines.

Table 2: rat sIPV-I type neutralization titer assay

Sample name	Test article 1	Sample 2	Test article 3	Control
					ED50 dilution factor	7.6	5.9	5.0	4.9

Table 3: rat sIPV-II type neutralization titer detection

Sample name	Test article 1	Sample 2	Test article 3	Control
					ED50 dilution factor	6.9	6.2	5.1	4.6

Table 4: rat sIPV-III type neutralization titer detection

Sample name	Test article 1	Sample 2	Test article 3	Control of
					ED50 dilution factor	3.9	3.2	2.4	1.9

Example 8

This example investigates the effect of DTaP-sIPV-Hib-AC combination vaccines with different aluminium content on immunogenicity.

Based on example 3, DTaP-sIPV-Hib-AC combination vaccines containing different aluminum concentrations of 0.2mg/ml, 0.4mg/ml, 0.6mg/ml, 0.8mg/ml, 1.0mg/ml, 1.2mg/ml and 1.4mg/ml were prepared, respectively, and subjected to in vivo efficacy tests for pertussis, diphtheria, tetanus, hib antibody positive conversion rate, A and C antibody positive conversion rate and sIPV, respectively, to analyze the immunogenicity of the combination vaccines. Pertussis, diphtheria, tetanus, hib and AC efficacy tests were performed according to the method for detecting the potency of pertussis, hib and AC in the third part of the "Chinese pharmacopoeia" 2015 edition. The method comprises the following steps of immunizing wistar rats respectively by adopting 3 dilutions of original times, 1/3 times and 1/9 times, injecting 10 mice in each group, collecting blood after immunization for 21 days, separating serum, detecting the titer of neutralizing antibodies by a cell method, and calculating the ED50 value. The results of antibody titers for diphtheria, tetanus, pertussis, hib and AC are shown in table 5, and the results of neutralizing titers for sipv antibodies are shown in table 6. The result shows that when the aluminum content in the DTaP-sIPV-Hib-AC combined vaccine is 0.2mg/ml, the diphtheria, tetanus and pertussis titers do not meet the standard; when the aluminum content is 0.4-1.2 mg/ml, the immune titer of each antigen meets the quality standard, and when the aluminum content is continuously increased, the titer of the vaccine tends to be reduced.

Table 5: combination vaccine diphtheria, tetanus, pertussis, hib and AC Titer outcomes

Table 6: sIPV potency outcomes in combination vaccines

Example 9

This example investigates the effect of M199 medium on the stability of combination vaccines.

Specifically, in this example, M199 medium was added to the mixed sIPV adsorption solution of the type I, type II and type III Sabin strains prepared in example 5 at different concentrations, and the effect on the stability of the virus antigen was examined. Different volumes of M199 medium concentrates (10 × M199 medium, i.e. concentrated M199 medium with all components having final concentrations 10 times of conventional M199 medium) were added, a control group without M199 addition and a test group with 5%, 10% and 20% (volume percentage) of M199 concentrates added to each 0.5ml of sIPV adsorption solution were set, and sIPV adsorption solutions having different concentrations of M199 were placed at 37 ℃, and the D antigen content in the sIPV adsorption solution was measured for 0 day, 7 days and 14 days, respectively. The detection results of the type I, type II and type III D antigen contents of the sIPV are respectively shown in fig. 1, fig. 2 and fig. 3, when M199 is not added, the D antigen content is continuously reduced along with the extension of the standing time, and when the sIPV adsorption solution is placed at 37 ℃ for 14 days, the D antigen reduction range in the poliovirus type I, type II and type III stock solutions is between 20% and 30%. When the addition amount of concentrated M199 in the IPV adsorption solution is 5%, 10% and 20%, the contents of IPV D antigens of I type, II type and III type are basically stable within 0-1 day, and after the sIPV adsorption solution is placed for 14 days at 37 ℃, the contents of the D antigens of I type, II type and III type are not obviously reduced, and the result shows that the addition of the M199 culture medium has an important effect on the stability of the poliovirus antigen.

Example 10

This example provides a method of testing the finished liquid vaccine formulations provided in examples 1-4.

1 identification test

The detection of PT, FHA and PRN antigens by enzyme-linked immunosorbent assay should contain corresponding antigens (appendix of Baibaishao combined vaccine adsorption in the three parts of '2015 edition' of Chinese pharmacopoeia). By adopting the immune double diffusion method (the third part of the 'Chinese pharmacopoeia' 2015 edition), the product should form an obvious precipitation line with diphtheria toxoid, tetanus toxoid, A-group meningococcal immune serum, C-group meningococcal immune serum, B-type haemophilus influenzae immune serum and tetanus toxoid immune serum.

2 physical examination

2.1 appearance: after shaking, the mixture should be in the form of uniform milky white suspension without non-scattering clots or foreign bodies

2.2 loading: checking according to law, and the content should not be less than the indicated amount (not less than 0.5 ml).

3 chemical examination

3.1 pH value: should be between 5.8 and 7.2.

3.2 aluminum content: should not be higher than 0.8 mg/dose.

3.3 free Formaldehyde content: should not be higher than 0.2g/L.

3.4 glutaraldehyde content: should be less than 0.01g/L.

3.5 polysaccharide content: taking D ribose as a reference, calculating the content of polysaccharide in the test sample according to the content of ribose, and using the dosage of 10-15 mu g for 1 time.

4 potency assay

4.1 acellular pertussis vaccine: the dilution is carried out to the first immunization dose by using a proper dilution factor, and then the dilution is carried out according to a 5-fold series. The immunization time was 21 days. The immune titer of the dose for 1 time of human use is not less than 4.0IU, and the lower limit of 95 percent of confidence limit is not less than 2.0IU. If the above requirements are not met, a retest can be performed, but all valid test results must be calculated as geometric means (e.g., using weighted geometric means when using probability analysis). If the requirements are met, the product is judged to be qualified.

4.2 diphtheria vaccine: the immune titer of diphtheria toxoid in each 1 human dose is not less than 30IU.

4.3 tetanus vaccine: the immunization titer of the tetanus toxoid in each 1 human dose should be not less than 40IU.

5 efficacy test

5.1 Haemophilus influenzae type b conjugate vaccine

Each batch of vaccine was injected subcutaneously with 10 NIH (or BALB/c) mice weighing 12-14 g, and the same batch of 10 mice was taken as a control and injected with 0.85% sodium chloride solution. Injecting Hib conjugate vaccine containing 2.5 microgram polysaccharide subcutaneously twice on the 1 st and 14 th days, collecting blood via retroorbital vein on the 21 st to 28 th days, measuring anti-Hib IgG antibody by ELISA method, and calculating Cutoff value by using absorbance value of mouse serum of 0.85% sodium chloride solution control group, wherein the vaccine group should have over 80% mouse serum anti-Hib IgG antibody level higher than Cutoff value.

5.2 Group A and group C meningococcal polysaccharide conjugate vaccine

Each batch of vaccine was injected subcutaneously with 10 NIH (or BALB/c) mice weighing 12-14 g, and the same batch of 10 mice was taken as a control and injected with 0.85% sodium chloride solution. Injecting the mixture twice subcutaneously on the 0 th day and the 14 th day, wherein the injection dose respectively comprises 2.5 mu g of polysaccharide of the A group and the C group, collecting blood on the 21 st to 28 th days after the 1 st needle, collecting blood by using Hib conjugate vaccine containing 2.5 mu g of polysaccharide in the injection dose, collecting blood by using retroorbital vein on the 21 st to 28 th days, measuring IgG antibody titer of the anti-A group and the anti-C group polysaccharide in blood cytoplasm by using an ELISA method, and calculating the Cutoff value by using the absorbance value of serum of a physiological sodium chloride solution control group mouse. The positive conversion rate of the antibody in the vaccine group is not less than 80%.

5.3 adsorption of Sabin Strain inactivated poliomyelitis vaccine

After serial gradient multiple dilution of each batch of vaccine, wistar rats with the cleaning grade above 180-220 g are immunized respectively, each group has 10 dilution degrees, half male and half female, and each animal is immunized with 0.5ml of test sample. Collecting blood for 21 days, separating serum, and storing at-20 deg.C. And (3) respectively detecting the titer of the neutralizing antibody of the anti-I/II/III type 3 viruses in the serum, and calculating the seroconversion rate. The efficacy test of the internal reference substance is carried out by the same method. The test article should be comparable in immunogenicity to the internal reference, i.e.: test article ED ₅₀ The value is not higher than 400% of the reference.

6, sterility inspection: the bacteria should be grown aseptically according to the examination by law.

7 pyrogen examination

According to legal examination, 1.0 mu g of polysaccharide is injected into the injection dose of lkg of the rabbit body weight, and the body temperature rise of three preliminarily tested rabbits is lower than 0.6 ℃, and the body temperature rise of 3 rabbits is comprehensively lower than 1.3 ℃; or in 5 re-tested rabbits, the temperature of the rabbits with the temperature rise of 0.6 ℃ or higher than 0.6 ℃ does not exceed 1 rabbit, and the total temperature rise of 8 rabbits in the initial test and the re-test is 3.5 ℃ or lower than 3.5 ℃, which all judge that the pyrogen examination of the test sample meets the regulation.

8 specific toxicity test

8.1 acellular pertussis vaccine: the toxic reference was diluted as indicated for each batch. NIH mice (female or male half) weighing 14-16 g were used, one for each dilution of the toxic reference and test article, and at least 10 for each group. Each mouse was injected with 0.5ml of the intraperitoneal solution, and the a-b tests were performed.

a) Mouse leukocytosis assay

Peripheral blood of the mice was collected 3 days after the injection to count leukocytes. The test result is statistically processed, and the leukocytosis activity of the mouse injected with the test article is not higher than 0.5LPU/ml.

b) Mouse histamine sensitization test

4 days after injection, 0.5ml of a solution (containing 4mg of histamine dihydrochloride or 2mg of histamine diphosphate) was intraperitoneally injected into each mouse, and 30 minutes later, the anal temperature of each mouse was measured. After the test result is treated by a statistical method, the activity of the sensitization toxicity of the histamine of the mouse of the test sample is not higher than 0.8HSU/ml, and no animal is dead.

8.2 diphtheria, tetanus vaccines

Using 250-350 g guinea pigs, each batch of the product was injected subcutaneously into the abdomen for 2.5ml, and injected bilaterally for 1.25ml, and observed for 30 days. The injection site can be infiltrated and become hard after 5-10 days, and may not be completely absorbed in 30 days. Weighing body weight on day 10, day 20 and day 30, increasing body weight before injection, and treating patients without topical suppuration, necrosis, tetanus and late stage paralysis.

9 toxicity reversal experiments: each test batch was placed at 37 ℃ for 4 weeks, following protocol 8.1 b).

10 bacterial endotoxin test: according to the general rule 1143 of three parts of the Chinese pharmacopoeia, the dose should be no higher than 100 EU.

The results of the measurements are shown in Table 7.

Table 7: vaccine test results

Example 11

This example provides a method of mixed immunization using the liquid vaccine formulations provided by the present invention. The method specifically comprises the following steps:

the liquid vaccine formulation provided in example 4 was used as a base immunization formulation vaccine and injected subcutaneously twice on days 1 and 14, respectively. Blood was collected from the orbit on day 28, and the IgG antibody titer in the mouse serum was measured by indirect ELISA. After 3 months (i.e., day 90), blood was collected again to assay the IgG titer in the serum, and the liquid vaccine preparation provided in example 1 was injected subcutaneously as a booster, and the IgG titer in the serum was assayed at 14 days (i.e., day 104) after the booster.

Experimental results show that the two immunizations with the liquid vaccine formulation provided in example 4 as the basic immunizing agent induced mice to produce significant neutralizing antibodies against poliovirus type I/II/III, antibodies against pertussis components (PT, FHA, PRN) (IgG), antibodies against DT (IgG), antibodies against TT (IgG), antibodies against Hib (IgG), antibodies against neisseria meningitidis a polysaccharide (IgG) and antibodies against neisseria meningitidis C polysaccharide (IgG). After 3 months of antibody levels in mice were tested and we found significant reductions in neutralizing antibodies to poliovirus type I/II/III, antibodies against pertussis components (PT, PRN) (IgG), antibodies against DT (IgG), antibodies against neisseria meningitidis polysaccharide group C (IgG). However, after immunization once with the liquid vaccine formulation provided in example 1 as a booster, the neutralizing antibodies against poliovirus type I/II/III, antibodies against pertussis components (PT, FHA, PRN) (IgG), antibodies against DT (IgG), antibodies against TT (IgG), antibodies against Hib (IgG), antibodies against neisseria meningitidis polysaccharide group a (IgG) and antibodies against neisseria meningitidis polysaccharide group C (IgG) were significantly improved in mice.

The results of the above assay for neutralizing antibody titers against type I/II/III sIPV are shown in FIG. 4.

The results of the above assay for antibody titer against pertussis (aP) are shown in fig. 5.

The results of the above assay for antibody titers against Diphtheria (DT) and tetanus (TT) are shown in FIG. 6.

The results of the above assays for antibody titers against Hib and Group A (Group A) Group C (Group C) Neisseria meningitidis are shown in FIG. 7.

In each of the above figures, P is < 0.05; * P < 0.01; * Represents P < 0.001.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A liquid vaccine composition, wherein the composition is a liquid and wherein a single dose of antigen per 0.5ml of the composition consists of: diphtheria toxoid 2-12.5 Lf, tetanus toxoid 2.5-5 Lf, pertussis toxin 20-25 mug, filamentous hemagglutinin 20-25 mug, pertussis adhesion 3-8 mug, inactivated Sabin I strain poliovirus 4-15 DU, inactivated Sabin II strain poliovirus 11-45 DU, inactivated Sabin III strain poliovirus 11-45DU, haemophilus influenzae type b capsular polysaccharide-protein conjugate 1.0-15 mug, meningococcus group A capsular polysaccharide-protein conjugate 1.0-15 mug, meningococcus group C capsular polysaccharide-protein conjugate 1.0-15 mug; or

The antigen in a single dose of the composition per 0.5ml consists of: diphtheria toxoid 2-12.5 Lf, tetanus toxoid 2.5-5 Lf, pertussis toxin 2.5-8 mug, filamentous hemagglutinin 5-8 mug, pertussis adhesion 2.5-3 mug, inactivated Sabin I strain poliovirus 4-15 DU, inactivated Sabin II strain poliovirus 11-45 DU, inactivated Sabin III strain poliovirus 11-45DU, haemophilus influenzae type b capsular polysaccharide-protein conjugate 1.0-15 mug, meningococcus group A capsular polysaccharide-protein conjugate 1.0-15 mug and meningococcus group C capsular polysaccharide-protein conjugate 1.0-15 mug.

2. The composition of claim 1, wherein the inactivated Sabin type I strain poliovirus, inactivated Sabin type II strain poliovirus and inactivated Sabin type III strain poliovirus in the composition have an antigen ratio of 1: (2.5-3.5): (2.5-3.5).

3. The composition of claim 1, wherein the haemophilus influenzae type b capsular polysaccharide-protein conjugate is formed by conjugating haemophilus influenzae type b capsular polysaccharide with a physiologically acceptable carrier protein; the physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, a nontoxic variant of diphtheria toxin CRM197, tetanus toxoid, meningococcal outer membrane protein;

and/or, the meningococcus capsular polysaccharide-protein conjugate is formed by conjugating meningococcus capsular polysaccharide with a physiologically acceptable carrier protein; the physiologically acceptable carrier protein is selected from one or more of diphtheria toxoid, nontoxic variant of diphtheria toxin CRM197, tetanus toxoid, meningococcal outer membrane protein.

4. The composition according to any one of claims 1 to 3, wherein the composition further comprises one or more of an aluminum adjuvant, a soluble phosphate buffer, sodium chloride, and a stabilizer.

5. The composition of claim 4, wherein the stabilizer is selected from one or more of vitamins, amino acids, and M199 medium.

6. A process for preparing a liquid vaccine composition according to any one of claims 1 to 5, comprising the steps of: mixing liquid raw materials including pertussis toxin, filamentous hemagglutinin, pertactin, diphtheria toxoid raw liquid, tetanus toxoid raw liquid, inactivated Sabin strain I poliovirus raw liquid, inactivated Sabin strain II poliovirus raw liquid, inactivated Sabin strain III poliovirus raw liquid, B haemophilus influenzae polysaccharide-protein conjugate raw liquid, A group meningococcus polysaccharide-protein conjugate raw liquid and C group meningococcus polysaccharide-protein conjugate raw liquid.

7. Use of a liquid vaccine composition according to any one of claims 1 to 5 for the preparation of a basic vaccine, wherein the pertussis toxin is 20 to 25 μ g, filamentous hemagglutinin is 20 to 25 μ g, and pertactin is 3 to 8 μ g, per 0.5ml single dose of the composition.

8. Use of a liquid vaccine composition according to any one of claims 1 to 5 for the preparation of a booster vaccine, wherein the pertussis toxin is 2.5 to 8 μ g, filamentous haemagglutinin is 5 to 8 μ g, and pertactin is 2.5 to 3 μ g per 0.5ml single dose of the composition.

9. A vaccine kit comprising a first chamber and a second chamber; a first chamber containing a basic vaccine prepared from the liquid vaccine composition according to any one of claims 1 to 5; a second chamber containing a booster vaccine prepared from the liquid vaccine composition according to any one of claims 1 to 5;

or, a first chamber, a second chamber, and a third chamber; the first and second chambers each contain a base vaccine prepared from the liquid vaccine composition of any one of claims 1 to 5; a third compartment containing a booster vaccine prepared from the liquid vaccine composition of any one of claims 1 to 5;

or, a first chamber, a second chamber, a third chamber, and a fourth chamber; the first chamber, the second chamber and the third chamber each contain a base vaccine prepared from the liquid vaccine composition of any one of claims 1 to 5; a fourth compartment containing a booster vaccine prepared from the liquid vaccine composition according to any one of claims 1 to 5;

when the liquid vaccine composition is prepared into a basic immune vaccine, 20-25 mug of pertussis toxin, 20-25 mug of filamentous hemagglutinin and 3-8 mug of pertactin are contained in each 0.5ml single dose of the composition;

when the liquid vaccine composition is prepared into a booster vaccine, 2.5-8 mug of pertussis toxin, 5-8 mug of filamentous hemagglutinin and 2.5-3 mug of pertactin are contained in each 0.5ml single dose of the composition.