CN115992101B - Preparation method of influenza virus split vaccine stock solution - Google Patents
Preparation method of influenza virus split vaccine stock solution Download PDFInfo
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Abstract
The invention discloses a preparation method of an influenza virus split vaccine stock solution, which comprises the following steps: s1: regulating the pH value of the harvest liquid in the chick embryo allantoic harvest liquid to be 5.5-6.5; after low-temperature incubation, adding a filter aid and uniformly stirring; filtering, collecting filtrate, adjusting pH to 7.0-8.0, standing at room temperature, stirring, concentrating, and washing to obtain concentrated solution; s2: the concentrated solution is purified by ultracentrifugation, and after ultrafiltration and washing for multiple times, sucrose is removed to obtain crude and pure virus solution; s3: adding an inactivating agent into the crude and pure virus liquid according to a proportion, and performing molecular sieve chromatography to obtain the refined and pure virus liquid; s4: adding a cracking agent into the refined virus liquid for cracking treatment, removing the cracking agent by ultrafiltration washing and filtering, and further concentrating to obtain influenza virus cracked vaccine stock solution. The preparation method solves the problems of low overall recovery rate, low impurity protein transmittance and poor virus purity in the preparation process of the influenza virus vaccine stock solution.
Description
Technical Field
The invention relates to the field of influenza virus vaccines, in particular to a preparation method of an influenza virus split vaccine stock solution.
Background
Influenza viruses, abbreviated influenza viruses, are enveloped RNA viruses, belonging to the orthomyxoviridae family, and are transmitted mainly by droplets when humans talk, by contact with patients suffering from influenza, or by contact with articles contaminated with influenza viruses. Influenza viruses include human influenza viruses and animal influenza viruses, and human influenza viruses can be classified into three types of influenza A (A), B (B) and C (C) according to antigenicity of viral nucleoprotein, and are all causative agents of influenza. Cough and fever are often fundamental features of influenza during influenza progression, whether adult or pediatric.
Influenza viruses are highly susceptible to mutation, which can lead to recurrent epidemics. When new strains appear, the human immunity level is reduced, and the WHO recommends influenza virus strains for vaccine production in the next year according to the variation rule of influenza viruses every year, so that people are necessary to vaccinate influenza vaccines every year.
In China, influenza vaccine (trivalent split vaccine of type A1, type A3 and type B) is prepared by growing influenza virus in chick embryo allantoic cavity, and after virus is inoculated in chick embryo allantoic cavity by chick embryo method, a large amount of urate can be generated along with embryo development and mass propagation of virus in chick embryo allantoic cavity culture process. The urate is not easy to dissolve, and is often aggregated into filiform or small-particle substances in chick embryo allantoic fluid, thus greatly affecting the purification of viruses and further affecting the batch-to-batch stability of stock solution preparation.
The existing method for removing the urate in the chick embryo allantoic harvest mainly comprises the steps of clarifying the chick embryo allantoic harvest by adopting a filtering or centrifuging mode, and removing the urate by adopting an ultrafiltration washing mode. The urate precipitate is suspended in the harvest liquid and is easily broken into small-fragment particles. If the filtration method is directly adopted for clarification treatment, the filtration pores are easily blocked in the filtration clarification treatment process, so that the virus loss rate is overlarge. And the centrifugal mode is adopted, so that the centrifugal speed is too low, and small particulate matters cannot be centrifuged down. And the too large centrifugal rate can increase the loss rate of viruses, and the conditions of large batch-to-batch difference and poor quality stability are easily caused. In the test process, the clarified allantoic harvest liquid is found to still continuously separate out white urate precipitate in the ultrafiltration concentration process, so that membrane bags are blocked, and the impurity protein transmittance is low, the virus purity is poor and the recovery rate is low.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of an influenza virus split vaccine stock solution, which solves the problems of low overall recovery rate, low impurity protein transmittance and poor virus purity in the preparation process of the influenza virus split vaccine stock solution.
The invention provides a preparation method of an influenza virus split vaccine stock solution, which comprises the following steps:
s1: adding sodium dihydrogen phosphate solution into chick embryo allantoic harvester to regulate pH of the harvester to 5.5-6.5; after low-temperature incubation, adding a filter aid and uniformly stirring; the purpose of this filtration is to remove a significant amount of the urate; collecting filtrate by filtering, adding disodium hydrogen phosphate solution to adjust pH to 7.0-8.0, standing at room temperature, stirring, concentrating by ultrafiltration and filtering to obtain concentrated solution;
s2: the concentrated solution is purified by ultracentrifugation, and after ultrafiltration and washing for multiple times, sucrose is removed to obtain crude and pure virus solution;
s3: adding beta-propiolactone into the crude and pure virus liquid according to a proportion for inactivation, and performing molecular sieve chromatography to obtain refined and pure virus liquid;
s4: adding a cracking agent into the refined virus liquid for cracking treatment to crack the inactivated virus, removing the cracking agent by ultrafiltration washing and filtering, and further concentrating to obtain the influenza virus cracked vaccine stock solution.
The pH value adjusted in the first step in the S1 is lower than that in the prior art, and a weak acid environment is maintained, so that the separation of the uric acid salt is facilitated; the second step of adjusting the pH value of the virus is stable in the pH range of 7.0-8.0, so that the virus is adjusted to the range to reduce the loss of antigen, and the second step can theoretically use sodium carbonate or sodium bicarbonate, sodium hydroxide with the concentration of about 0.1M and other weak bases, but can introduce new impurities, which is not beneficial to the preparation of vaccines. The disodium hydrogen phosphate (weak alkaline) and the former sodium dihydrogen phosphate can be just neutralized to about pH7.5, and no new impurities are introduced. The S1 adopts low pH and low temperature incubation, and filter aid is added for filtration, so that urate in monovalent harvest liquid is removed, and the antigen recovery rate of vaccine preparation is greatly improved; the filter aid removes the uric acid salt and simultaneously adsorbs most of pigment and part of impurity protein, thereby improving the purity of the virus; the pH of the allantoic harvest liquid is adjusted by utilizing the characteristic of mild conditions of disodium hydrogen phosphate and sodium dihydrogen phosphate, so that the antigen loss can be reduced to the greatest extent, and other components are prevented from being introduced. Other weak acids may be used, such as acetic or hydrochloric acids at about 0.1M, but such acids introduce impurities and cause greater antigen losses than sodium dihydrogen phosphate. Inactivation is generally with formaldehyde or beta-propiolactone. The inactivation step can be replaced by formaldehyde, but the formaldehyde has low inactivation efficiency, high residue and high side effect on organisms, and is not used abroad. The beta-propiolactone is used for inactivation without residue, and the inactivation efficiency is high.
Further, the pH of the harvest liquid is adjusted to 6 by adding a sodium dihydrogen phosphate solution into the S1.
Further, the temperature of low-temperature incubation in S1 is-20 ℃, and the incubation is carried out for 60 minutes.
Further, disodium hydrogen phosphate solution is added into the S1 to adjust the pH value of the filtrate to 7.5.
Further, the dosage ratio of the harvest liquid to the filter aid in the step S1 is 1L:10g.
Further, the filter aid in the step S1 is any one of perlite, diatomite, talcum powder and activated carbon.
Further, the rotational speed of the ultracentrifugation in S2 is 25000rpm to 35000rpm for 2 to 4 hours.
Further, the operation of removing sucrose in S2 is: and (3) adopting a 300kD ultrafiltration membrane bag, washing and filtering 5 times by using PBS to obtain a desugared treatment solution, sampling and detecting the protein concentration, and diluting the protein by using PBS.
Further, in the step S3, beta-propiolactone is added into the crude and pure virus liquid according to a volume ratio of 1:4000.
Further, the cracking agent in the S4 is any one of Triton X-100, polysorbate 80, deoxysodium cholate, triton N-101 and cetyltrimethylammonium bromide.
In conclusion, compared with the prior art, the invention achieves the following technical effects:
1. by adopting the preparation method, the overall recovery rate of the stock solution preparation can reach more than 30%, and the overall recovery rate of the traditional method is less than 20%.
2. By adopting the preparation method, the transmittance of the hybrid protein can reach more than 99.9 percent.
3. By adopting the preparation method of the invention, the purity of the virus can reach more than 1.87.
4. The low pH and low temperature incubation are adopted, diatomite is added for filtering, and uric acid salt in the monovalent harvest liquid is removed, so that the antigen recovery rate of vaccine preparation is greatly improved;
5. the diatomite absorbs most of pigments and part of impurity proteins while removing the urates, so that the purity of the virus is improved;
6. the pH of the allantoic harvest liquid is adjusted by utilizing the characteristic of mild conditions of disodium hydrogen phosphate and sodium dihydrogen phosphate, so that the antigen loss can be reduced to the greatest extent, and other components are prevented from being introduced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic flow chart of a preparation method according to the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
The invention provides a preparation method of an influenza virus split vaccine stock solution. According to the invention, a sodium dihydrogen phosphate solution is added into chick embryo allantoic harvest liquid to regulate the pH value of the harvest liquid to 5.5-6.5. After incubation at low temperature, perlite or diatomite is added in proportion and stirred uniformly. Collecting filtrate by filtration, adding disodium hydrogen phosphate solution to adjust pH to 7.0-8.0, standing at room temperature, stirring for a certain time, concentrating by ultrafiltration and filtering to obtain concentrated solution. And (3) performing super-separation purification on the concentrated solution, performing ultrafiltration washing filtration for multiple times, and removing sucrose in the concentrated solution to obtain crude and pure virus solution. Adding beta-propiolactone into crude and pure virus liquid according to a certain proportion, inactivating, and performing molecular sieve chromatography according to 3-5% of sample loading quantity to obtain the refined and pure virus liquid. Adding a cracking agent into the refined virus liquid for cracking treatment, removing the cracking agent by ultrafiltration washing and filtering, and further concentrating to obtain influenza virus cracked vaccine stock solution.
The invention aims at realizing the following steps: the preparation process of split influenza virus vaccine stock solution includes inoculation and culture, harvesting, clarifying, concentration, purification, inactivation, splitting, secondary purification to obtain stock solution, and the key is that the virus harvest liquid is concentrated, washed, purified, inactivated, split and secondary purified after the great amount of uric acid salt is eliminated. By adopting the method, the overall recovery rate of the stock solution preparation can reach more than 30 percent (the overall recovery rate of the traditional method is less than 20 percent).
The method for removing the urates comprises the following steps: sodium dihydrogen phosphate or potassium dihydrogen phosphate of 0.2. 0.2M is added into allantoic fluid under stirring until the pH of allantoic fluid is 5.5-6.5. Standing the obtained solution at-20deg.C for 60-90 min, adding diatomite according to 8-12g/L allantoic fluid, and stirring. Clarifying with 5-20 μm filter element, adding 0.2M disodium hydrogen phosphate or dipotassium hydrogen phosphate under stirring until pH of clarified solution is 7.0-8.0, and stirring at room temperature overnight to obtain virus clarified solution with removing urate.
Examples
The embodiment discloses a preparation method of an influenza virus split vaccine stock solution, which specifically comprises the following steps:
(1) Adjusting the pH value of the chick embryo allantoic harvest liquid: adjusting the pH of the 30L harvest liquid to 6.0 by using a 0.1 mol/L sodium dihydrogen phosphate solution;
(2) Low temperature incubation: standing the adjusted harvest liquid at the temperature of minus 20 ℃ for 60 minutes;
(3) pH adjustment, filtration and clarification: diatomite 300 g was added at 10g/L, and after stirring well, the pH was adjusted to 7.5 with 0.1 mol/L disodium hydrogen phosphate. Clarifying and filtering with 5.0 μm filter core, and the transmembrane pressure should not exceed 0.1 Mpa;
(4) Concentrating: ultrafiltration and concentration are carried out for 10 times by using a 300kD ultrafiltration membrane bag, and washing and filtering are carried out for 5 times by using 10mmol/L PBS (pH 7.2), so as to obtain concentrated solution;
(5) And (3) super-separation purification: using ultracentrifuge zone centrifugation, 500 ml 10mmol/L PBS (pH 7.2) was poured directly into the zone rotor chamber, the top cover was closed, and 55% sucrose (10 mmol/L, pH 7.2) was pumped from the side well at a flow rate of 50 ml/min until the liquid was drained from the middle well. Transferring to a mesopore for loading, pumping into concentrated solution 600 ml at a flow rate of 25 ml/min, and further pumping into the mesopore with the PBS of 50 ml for sealing. The centrifugation speed was 30000 rpm, the temperature was 15℃and the centrifugation time was 3 h. After centrifugation, 55% sucrose (10 mmol/L, pH 7.2) was pumped from the side wells at a flow rate of 50 ml/min and the viral peaks were collected by UV and conductivity monitoring;
(6) Desugaring treatment (sucrose removal): filtering with 300kD ultrafiltration membrane bag, washing with 10mmol/L PBS (pH 7.2) for 5 times to obtain desugared treating solution 520ml, sampling to detect protein concentration, and diluting with 10mmol/L PBS (pH 7.2) to 500-1000 μg/ml;
(7) And (3) inactivation treatment: adding beta-propiolactone according to a ratio of 1:4000, standing at 2-8 ℃, oscillating for 24-25 hours, and then placing in a 37 ℃ water bath 2 h;
(8) And (3) chromatographic purification: the molecular sieve gel medium sepharose6FF and the chromatographic equipment AKTA PILOT are adopted for chromatographic purification, and the buffer solution is 10mmol/L PBS (pH 7.2). The loading flow rate was 60 cm/h. After balancing 2-3 column volumes, the inactivating treatment solution is sampled according to 5%, molecular sieve chromatography purification is carried out, the eluting flow rate is 30 cm/h, and the chromatography purification solution is obtained after collection.
(9) And (3) cracking treatment: adding a cracking agent TritonX-100 according to the final concentration of 0.7%, and oscillating at 80 rpm for 60 minutes at 22-25 ℃ to obtain virus cracking treatment liquid;
(10) And (3) secondary purification: and (3) adopting a 30kD ultrafiltration membrane bag, washing and filtering by more than 40 times with 10mmol/L PBS (pH 7.2), concentrating by 10 times, and filtering and sterilizing by a 0.22 mu m filter to obtain the monovalent stock solution.
Comparative example traditional method for preparing influenza virus split vaccine stock solution specifically comprises the following steps:
(1) Clarifying and filtering the harvest liquid: clarifying and filtering 30L of harvest liquid directly with 5.0 μm filter element, and the transmembrane pressure is not more than 0.1 Mpa;
(2) Virus inactivation: 2% formaldehyde was added in a 1:100 ratio to give a final formaldehyde concentration of 200. Mu.g/ml. And (3) conveying the virus liquid into an inactivation tank, and inactivating for 60-68 hours at the temperature of 2-8 ℃ and the stirring speed of 10 Hz.
(3) Concentrating: ultrafiltration and concentration are carried out for 10 times by using a 300kD ultrafiltration membrane bag, and washing and filtering are carried out for 5 times by using 10mmol/L PBS (pH 7.2), so as to obtain concentrated solution;
(3) And (3) super-separation purification: using ultracentrifuge zone centrifugation, 500 ml of 10mmol/L PBS (pH 7.2) was poured directly into the zone rotor chamber, the top cover was closed, and 55% sucrose (10 mmol/L, pH 7.2) was pumped from the side well at a flow rate of 50 ml/min until the liquid was drained from the middle well. Transferring to a mesopore for loading, pumping into concentrated solution 600 ml at a flow rate of 25 ml/min, and further pumping into the mesopore with the PBS of 50 ml for sealing. The centrifugation speed was 30000 rpm, the temperature was 15℃and the centrifugation time was 3 h. After centrifugation, 55% sucrose (10 mmol/L, pH 7.2) was pumped from the side wells at a flow rate of 50 ml/min and the viral peaks were collected by UV and conductivity monitoring;
(4) Desugarization treatment: filtering with 300kD ultrafiltration membrane bag, washing with 10mmol/L PBS (pH 7.2) for 5 times to obtain desugared treatment solution 520ml, sampling to detect protein concentration, and diluting the protein concentration to 500-1000 μg/ml with 10mmol/L PBS (pH 7.2);
(5) And (3) chromatographic purification: the molecular sieve gel medium sepharose6FF and the chromatographic equipment AKTA PILOT are adopted for chromatographic purification, and the buffer solution is 10mmol/L PBS (pH 7.2). The loading flow rate was 60 cm/h. After balancing 2-3 column volumes, the inactivating treatment solution is sampled according to 5%, molecular sieve chromatography purification is carried out, the eluting flow rate is 30 cm/h, and the chromatography purification solution is obtained after collection.
(6) And (3) cracking treatment: adding a cracking agent TritonX-100 according to the final concentration of 0.7%, and oscillating at 80 rpm for 60 minutes at 22-25 ℃ to obtain virus cracking treatment liquid;
(7) And (3) secondary purification: and (3) adopting a 30kD ultrafiltration membrane bag, washing and filtering by more than 40 times with 10mmol/L PBS (pH 7.2), concentrating by 10 times, and filtering and sterilizing by a 0.22 mu m filter to obtain the monovalent stock solution.
The total recovery rate of hemagglutinin, the transmittance of hetero-protein and the purity of virus in each step of the above examples and comparative examples were measured, respectively, and the total recovery rate was obtained by measuring the content of antigen (i.e., hemagglutinin) before and after the comparative process. Protein permeation rate: the protein content was measured by lowry method (2020 edition, second method of Chinese pharmacopoeia, general rule 0731). The protein permeation rate= [1- (volume of dope protein content)/(volume of harvest protein content) ]: 100%; the viral purity was calculated as viral purity = stock protein content/stock hemagglutinin content. The method for measuring the protein content of the stock solution adopts a lowry method; the hemagglutinin content is determined by a unidirectional immunodiffusion method; overall recovery = (stock volume x hemagglutinin content)/(harvest volume x hemagglutinin content) ×100. The results are shown in tables 1 and 2, respectively.
TABLE 1 prior art method
TABLE 2 method of the invention
The results show that the method removes the urates through the pretreatment of earlier clarification, and the virus stock solution prepared through the beta-propiolactone inactivation method improves the recovery rate of the preparation of the influenza virus split vaccine stock solution, and improves the impurity protein transmittance and the virus purity. The virus purity is the ratio of protein content/hemagglutinin content, the higher the hemagglutinin content, the lower the protein content, the better the purity, so the smaller the value, the higher the virus purity.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (3)
1. The preparation method of the influenza virus split vaccine stock solution is characterized by comprising the following steps:
s1: adding sodium dihydrogen phosphate solution into chick embryo allantoic harvester to regulate pH of the harvester to 6.0; standing the adjusted harvest liquid at-20 ℃ for 60 minutes, adding diatomite according to 10g/L, and uniformly stirring; collecting filtrate by adopting a filtering mode, adding disodium hydrogen phosphate solution to adjust the pH of the filtrate to 7.5, filtering, concentrating by adopting an ultrafiltration concentration method, and washing and filtering to obtain concentrated solution;
s2: purifying the concentrated solution by ultracentrifugation, wherein the temperature of the centrifugation is 15 ℃, and removing sucrose after ultrafiltration and washing filtration for 5 times to obtain crude and pure virus solution;
s3: adding beta-propiolactone into the crude and pure virus liquid according to the volume ratio of 1:4000 for inactivation, and performing molecular sieve chromatography to obtain the refined and pure virus liquid;
s4: adding TritonX-100 into the purified virus solution for cracking treatment, adding TritonX-100 as a cracking agent according to the final concentration of 0.7%, removing TritonX-100 by ultrafiltration washing and filtering, and further concentrating to obtain influenza virus cracked vaccine stock solution.
2. The method according to claim 1, wherein the ultracentrifugation in step S2 is performed at a rotational speed of 25000rpm to 35000rpm for 2 to 4 hours.
3. The method according to claim 1, wherein the sucrose removal operation in step S2 is: and (3) adopting a 300kD ultrafiltration membrane bag, washing and filtering 5 times by using PBS to obtain a desugared treatment solution, sampling and detecting the protein concentration, and diluting the protein by using PBS.
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| WO2022009121A1 (en) * | 2020-07-07 | 2022-01-13 | Spicona Inc. | Sars-cov-2 and influenza combination vaccine |
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