Preparation method of high-dose influenza virus split vaccine stock solution

Abstract

The invention relates to the field of biological pharmacy, in particular to a preparation method of a high-dose influenza virus split vaccine stock solution. The method comprises the following steps: carrying out first sucrose density gradient centrifugation on an influenza virus concentrated solution, and collecting a centrifugate with a sugar degree interval of 28-50% to obtain a first influenza virus centrifugate; performing second sucrose density gradient centrifugation on the first influenza virus centrifugate, and respectively collecting centrifugates with the sugar degree ranges of 35%-50% and 22%-35% to obtain a second influenza virus centrifugate and a third influenza virus centrifugate; and carrying out third sucrose density gradient centrifugation on the third influenza virus centrifugate, and collecting the centrifugate with the sugar degree interval of 35-50% to obtain a fourth influenza virus centrifugate. The preparation method can reduce the content of ovalbumin, optimize the proportion of protein hemagglutinin, improve the recovery rate of hemagglutinin, efficiently remove impurities, simplify the purification process, and improve the safety, immune effect, stability and production efficiency of influenza vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and more particularly, to a method for preparing a high-dose inactivated influenza virus vaccine bulk. Background Art

[0002] Influenza viruses are RNA viruses belonging to the Orthomyxoviridae family. Their envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are key targets for vaccine design.

[0003] Currently, the inactivated influenza virus vaccines on the market generally use a conventional dose (15 μg HA / strain / dose). Most of their production processes are based on one or two sucrose density gradient centrifugation methods and follow the standardized process of "inactivation first and then lysis". However, this technical solution has the following limitations:

[0004] 1) Insufficient immunogenicity: For the elderly (≥65 years old) and immunocompromised populations, the serum protection rate (SPR) and serum conversion rate (SCR) induced by the conventional dose vaccine are significantly lower than those of the healthy adult population, making it difficult to form an effective immune barrier.

[0005] 2) Bottleneck in dose increase: The inactivated influenza virus vaccines produced by the existing process have problems of high protein hemagglutination ratio and low sample purity. If the existing process is used to quadruple the HA content to produce a high-dose vaccine, the total protein content will increase significantly, leading to a higher frequency and greater severity of adverse reactions.

[0006] In summary, it is of great clinical application value and broad market prospects to prepare a high-dose inactivated influenza virus vaccine with high purity, high immunogenicity, good safety, and meeting the higher requirements for vaccine immune protection of the elderly population and others.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] To solve the defects and deficiencies existing in the existing influenza vaccine production technology, the present invention proposes a method for preparing a high-dose inactivated influenza virus vaccine bulk. On the basis of the existing technology, by optimizing the production process, this method can produce a monovalent influenza virus bulk with lower impurity content and higher purity.

[0009] To achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0010] One aspect of the present invention relates to a method for preparing a high-dose inactivated influenza virus vaccine bulk, comprising the following steps:

[0011] (a) Concentrate the influenza virus liquid by performing the first sucrose density gradient centrifugation, collect the centrifugate with a sugar concentration range of 28% - 50% to obtain the first influenza virus centrifugate; perform the second sucrose density gradient centrifugation on the first influenza virus centrifugate, and respectively collect the centrifugates with sugar concentration ranges of 35% - 50% and 22% - 35% to obtain the second influenza virus centrifugate and the third influenza virus centrifugate;

[0012] (b) Perform the third sucrose density gradient centrifugation on the third influenza virus centrifugate, collect the centrifugate with a sugar concentration range of 35% - 50% to obtain the fourth influenza virus centrifugate;

[0013] (c) Mix and dilute the second influenza virus centrifugate and the fourth influenza virus centrifugate to perform the first washing and filtration to obtain the influenza virus de - sugared and purified liquid;

[0014] (d) Perform the first ultrasonic treatment on the influenza virus de - sugared and purified liquid, filter it and then perform lysis to obtain the influenza virus lysate; perform the second washing and filtration to remove the lysing agent in the influenza virus lysate to obtain the influenza virus lysate purified liquid;

[0015] (e) Add an inactivating agent to the influenza virus lysate purified liquid for inactivation to obtain the influenza virus inactivated liquid; perform the third washing and filtration to remove the inactivating agent in the influenza virus inactivated liquid to obtain the influenza virus inactivated purified liquid; perform the second ultrasonic treatment and sterile filtration on the influenza virus inactivated purified liquid to obtain the monovalent influenza virus stock solution.

[0016] The preparation method of the high - dose influenza virus split vaccine stock solution can significantly reduce the ovalbumin content, optimize the proportion of protein hemagglutinin, improve the hemagglutinin recovery rate, efficiently remove impurities, simplify the purification process, thereby improving the safety, immune effect, stability and production efficiency of the influenza vaccine, and reducing the production cost, having significant technical advantages and broad application prospects.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) Significantly reduce the ovalbumin content and improve the vaccine safety: The present invention significantly reduces the ovalbumin content in the monovalent influenza virus stock solution. The average content of each type is only 3.9 ng / ml, which is 92.9% lower than the average ovalbumin content of 55.1 ng / ml in the monovalent stock solution prepared by the prior art. At the same time, the present invention eliminates the difference in ovalbumin content among different types, improves the uniformity of product quality, thereby significantly reducing the potential risk of allergic reactions and improving the safety of the vaccine.

[0019] (2) Optimize the proportion of protein hemagglutinin to enhance the immune effect of the vaccine: The average value of the ratio of protein content to hemagglutinin content in the monovalent stock solution of each type of influenza virus prepared by the present invention is 1.8, which is much lower than the average value of 3.1 in the prior art and also much lower than the standard (≤4.5) specified in the pharmacopoeia for influenza virus split vaccines. A lower protein hemagglutinin ratio means that a higher proportion of the effective antigen (hemagglutinin) is contained in the vaccine per unit dose, which theoretically can enhance the immunogenicity of the vaccine and improve the immune effect of the vaccine.

[0020] (3) Optimize the splitting and inactivating processes to improve the stability and production efficiency of the vaccine: The present invention adopts a production process of splitting first and then inactivating, effectively solving the problem that the virus is prone to aggregation after adding the inactivating agent in the existing process, thereby improving the degree and uniformity of splitting, enhancing the splitting effect and stability of the vaccine. At the same time, after the virus is split, the exposed area of the virus particles increases, which can make the inactivating effect better while improving the safety of the vaccine, and at the same time shortens the time required for inactivation, significantly improves the production efficiency, and reduces the production cost.

[0021] (4) Improve the hemagglutinin recovery rate, reduce the protein hemagglutinin ratio, and improve the product quality: Through the multiple gradient density centrifugation process, the hemagglutinin recovery rate is significantly improved. The average hemagglutinin recovery rate of the deglycosylated and purified solution of each type of influenza virus prepared by the present invention reaches 93%, which is 35% higher than 58% of the existing process. At the same time, the average value of the protein hemagglutinin ratio for each type also decreases from 4.1 in the existing process to 2.6, further improving the quality and purity of the product.

[0022] (5) Efficiently remove impurities and improve the purity of the vaccine: The present invention uses a 10kD ultrafiltration membrane package to ultrafilter and purify the inactivated virus solution, which can effectively remove impurities such as formaldehyde, and can further remove smaller pollutants such as endotoxin and small molecule impurities. By optimizing the membrane area and process parameters, the efficiency and yield of the entire purification process are ensured, thereby realizing the purification of the virus solution with high efficiency and high purity, and improving the safety of the vaccine.

[0023] (6) Simplify the purification process, reduce the production cost, and improve the process flexibility: The present invention abandons the commonly used chromatography purification step in the traditional process and adopts the ultrafiltration method for purification treatment throughout the process. This innovative improvement not only significantly improves the recovery rate of the active ingredients, but also greatly simplifies the operation process, making the entire process more efficient and convenient. At the same time, the present invention performs well in cost control, significantly reducing the production cost compared with the traditional method, and also has more advantages in terms of process flexibility and controllability, and can better adapt to different production scales and purification requirements. Detailed implementation manners

[0024] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, those skilled in the art will understand that the following described embodiments are some embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0025] One aspect of the present invention relates to a method for preparing a high-dose split influenza virus vaccine bulk, comprising the following steps:

[0026] (a) Subject the influenza virus concentrate to a first sucrose density gradient centrifugation, and collect the centrifugate with a sugar density range of 28% - 50% (for example, it can be, but is not limited to, the point value of any one of 28%, 30%, 33%, 35%, 38%, 40%, 43%, 45%, 48% or 50% or the range value between any two of them) to obtain a first influenza virus centrifugate; subject the first influenza virus centrifugate to a second sucrose density gradient centrifugation, and respectively collect the centrifugates with sugar density ranges of 35% - 50% (for example, it can be, but is not limited to, the point value of any one of 35%, 38%, 40%, 43%, 45%, 48% or 50% or the range value between any two of them) and 22% - 35% (for example, it can be, but is not limited to, the point value of any one of 22%, 25%, 28%, 30%, 33% or 35% or the range value between any two of them) to obtain a second influenza virus centrifugate and a third influenza virus centrifugate;

[0027] (b) Subject the third influenza virus centrifugate to a third sucrose density gradient centrifugation, and collect the centrifugate with a sugar density range of 35% - 50% (for example, it can be, but is not limited to, the point value of any one of 35%, 38%, 40%, 43%, 45%, 48% or 50% or the range value between any two of them) to obtain a fourth influenza virus centrifugate;

[0028] (c) Mix the second influenza virus centrifugate and the fourth influenza virus centrifugate, and then perform dilution and a first washing and filtration to obtain a de-sugared and purified influenza virus solution;

[0029] (d) Subject the de-sugared and purified influenza virus solution to a first ultrasonic treatment, filtration, and then lysis to obtain an influenza virus lysate; perform a second washing and filtration to remove the lysing agent in the influenza virus lysate to obtain an influenza virus lysate purification solution;

[0030] (e) Inactivate the influenza virus lysis and purification solution by adding an inactivator to obtain an inactivated influenza virus solution; after the third washing and filtration to remove the inactivator from the inactivated influenza virus solution, an inactivated and purified influenza virus solution is obtained; perform a second ultrasonic treatment and sterilizing filtration on the inactivated and purified influenza virus solution to obtain a monovalent influenza virus stock solution.

[0031] The preparation method of the high-dose influenza virus split vaccine stock solution can significantly reduce the ovalbumin content, optimize the protein hemagglutinin ratio, increase the hemagglutinin recovery rate, efficiently remove impurities, simplify the purification process, thereby improving the safety, immune effect, stability and production efficiency of the influenza vaccine, and reducing the production cost, having significant technical advantages and broad application prospects.

[0032] The preparation method of the high-dose influenza virus split vaccine stock solution uses an eKII continuous flow ultracentrifuge to perform multiple centrifugal purification operations on the influenza virus sample. Its core principle is to utilize the difference in sedimentation rates of different substances in the centrifugal force field, combined with a sucrose density gradient, so that different components in the sample are distributed in different sucrose concentration regions during centrifugation, thereby realizing the separation and purification of the target component. Compared with the traditional method, this centrifugal purification process can effectively reduce the ovalbumin content, basically eliminate the difference in ovalbumin content among different serotypes, and at the same time increase the hemagglutinin recovery rate and the removal rate of miscellaneous proteins, making the stock solution purer.

[0033] Perform the first sucrose density gradient centrifugation on the influenza virus concentrate and collect the centrifugate in the sugar concentration range of 28% - 50% to preliminarily separate the virus particles from most of the impurities, laying a foundation for subsequent purification. The target virus and some impurities may be included in this range, with a relatively high recovery rate but limited purity.

[0034] Perform the second sucrose density gradient centrifugation on the first influenza virus centrifugate, and collect the centrifugates in two sugar concentration ranges of 35% - 50% and 22% - 35% respectively. The range of 35% - 50% is usually an enrichment area for intact viruses (with higher density), removing low-density impurities (such as empty-shell viruses, host proteins). The range of 22% - 35% may contain low-density impurities (such as virus fragments, nucleic acids) or virus subpopulations with lower density. The second sucrose density gradient centrifugation can separate virus subpopulations with different densities (such as intact viruses, empty-shell viruses, etc.), can more precisely recover the target virus, reduce the loss of the target virus, and improve the purity of virus particles.

[0035] Perform the third sucrose density gradient centrifugation on the third influenza virus centrifugate, and collect the centrifugate in the sugar concentration range of 35% - 50%, which can further remove low-density impurities (such as empty-shell viruses, virus fragments, etc.), and at the same time can recover the target virus that may remain therein, further increasing the recovery rate.

[0036] This process significantly improves the virus purity and recovery rate by performing multiple sucrose density gradient centrifugations and gradually optimizing the sugar concentration collection range. Meanwhile, it has a stronger ability to remove impurities and is particularly suitable for products with high requirements for virus purity and recovery rate.

[0037] The present invention adopts a process of first lysing and then inactivating. First, the sample is lysed. Through specific lysing agents and conditions, the outer shell structure of the virus is destroyed, enabling the components inside the virus to be fully exposed. At the same time, it avoids the problem in the traditional process of first inactivating and then lysing, where the virus particles are prone to aggregation after adding the inactivating agent, which affects the virus lysis effect. It improves the degree and uniformity of lysis, ensuring that the virus is completely lysed. Subsequently, the inactivation operation is carried out. Using appropriate inactivating agents and conditions, the virus is inactivated while retaining its immunogenicity. Compared with the traditional process, the process of first lysing and then inactivating of the present invention has multiple technical advantages. On the one hand, it improves the lysis effect and the stability of the stock solution. Sufficient lysis enables the antigen components of the virus to be better released and exposed, which is beneficial for subsequent immune responses. At the same time, the uniform lysis process ensures the consistency of each component in the stock solution, improving the quality stability of the product. On the other hand, this process reduces the inactivation time. Since the virus is more easily affected by the inactivating agent after lysis, the time required for inactivation can be shortened. Finally, while ensuring the safety of the vaccine, it not only improves the production efficiency, reduces the production cycle, but also reduces the production cost, including costs in aspects such as energy consumption and reagent use.

[0038] Further, the concentration of the sucrose solution for the first sucrose density gradient centrifugation is 50wt% - 60wt%, including but not limited to any point value among 50wt%, 52wt%, 54wt%, 56wt%, 58wt% or 60wt% or the range value between any two of them.

[0039] Further, the sample injection flow rate for the first sucrose density gradient centrifugation is 200 - 250 ml / min, including but not limited to any point value among 200 ml / min, 210 ml / min, 220 ml / min, 230 ml / min, 240 ml / min or 250 ml / min or the range value between any two of them.

[0040] Further, the speed of the first sucrose density gradient centrifugation is 34000 - 36000 rpm, including but not limited to any point value among 34000 rpm, 34200 rpm, 34400 rpm, 34600 rpm, 34800 rpm, 35000 rpm, 35200 rpm, 35400 rpm, 35600 rpm, 35800 rpm or 36000 rpm or the range value between any two of them.

[0041] Furthermore, the time for the first sucrose density gradient centrifugation is 40 to 50 min, including but not limited to the point values of any one of 40 min, 42 min, 44 min, 46 min, 48 min or 50 min, or the range values between any two of them.

[0042] By precisely controlling the concentration of the sucrose solution, the injection flow rate, the centrifugation speed and the time of the first sucrose density gradient centrifugation, the separation efficiency of virus particles from impurities can be significantly improved. The optimized centrifugation conditions contribute to more thoroughly removing impurities in virus particles, such as ovalbumin, etc., thereby improving the purity of virus particles. Improving the purity of virus particles helps reduce potential allergens, thereby enhancing the safety of the vaccine. The clear range of centrifugation parameters helps ensure the stability and consistency of vaccine quality between different batches.

[0043] Furthermore, the concentration of the sucrose solution for the second sucrose density gradient centrifugation is 50 wt% to 60 wt%, including but not limited to the point values of any one of 50 wt%, 52 wt%, 54 wt%, 56 wt%, 58 wt% or 60 wt%, or the range values between any two of them.

[0044] Furthermore, the injection flow rate for the second sucrose density gradient centrifugation is 200 to 250 ml / min, including but not limited to the point values of any one of 200 ml / min, 210 ml / min, 220 ml / min, 230 ml / min, 240 ml / min or 250 ml / min, or the range values between any two of them.

[0045] Furthermore, the speed of the second sucrose density gradient centrifugation is 34000 to 36000 rpm, including but not limited to the point values of any one of 34000 rpm, 34200 rpm, 34400 rpm, 34600 rpm, 34800 rpm, 35000 rpm, 35200 rpm, 35400 rpm, 35600 rpm, 35800 rpm or 36000 rpm, or the range values between any two of them.

[0046] Furthermore, the time for the second sucrose density gradient centrifugation is 40 to 50 min, including but not limited to the point values of any one of 40 min, 42 min, 44 min, 46 min, 48 min or 50 min, or the range values between any two of them.

[0047] By defining the technical parameters of the second sucrose density gradient centrifugation, residual impurities in the virus particles can be further removed, and the purity of the virus particles can be improved. The optimized centrifugation conditions help the virus particles to be more evenly distributed in the sucrose gradient, facilitating subsequent separation and purification. By increasing the purity and uniformity of the virus particles, the immune effect of the vaccine can be further enhanced.

[0048] Further, the concentration of the sucrose solution for the third sucrose density gradient centrifugation is 50wt% - 60wt%, including but not limited to any point value among 50wt%, 52wt%, 54wt%, 56wt%, 58wt% or 60wt% or the range value between any two of them.

[0049] Further, the injection flow rate for the third sucrose density gradient centrifugation is 200 - 250 ml / min, including but not limited to any point value among 200 ml / min, 210 ml / min, 220 ml / min, 230 ml / min, 240 ml / min or 250 ml / min or the range value between any two of them.

[0050] Further, the speed of the third sucrose density gradient centrifugation is 34000 - 36000 rpm, including but not limited to any point value among 34000 rpm, 34200 rpm, 34400 rpm, 34600 rpm, 34800 rpm, 35000 rpm, 35200 rpm, 35400 rpm, 35600 rpm, 35800 rpm or 36000 rpm or the range value between any two of them.

[0051] Further, the time of the third sucrose density gradient centrifugation is 40 - 50 min, including but not limited to any point value among 40 min, 42 min, 44 min, 46 min, 48 min or 50 min or the range value between any two of them.

[0052] By defining the technical parameters of the third sucrose density gradient centrifugation, impurities around the virus particles can be further removed while ensuring that the integrity of the virus particles is not damaged. The optimized centrifugation conditions help maintain the stability and activity of the virus particles, thereby improving the storage stability and shelf life of the vaccine, and ensuring that the produced vaccine meets the requirements of high quality, high safety and high immune effect.

[0053] Further, when collecting the first influenza virus centrifugate, the second influenza virus centrifugate, the third influenza virus centrifugate, and the fourth influenza virus centrifugate, adjust the flow rate to 90-110 mL / min, including but not limited to the point value of any one of 90 mL / min, 95 mL / min, 100 mL / min, 105 mL / min, or 110 mL / min or the range value between any two of them.

[0054] Further, the dilution multiple is 3-5 times.

[0055] Further, the first washing and filtration includes: washing and filtration with equal volume.

[0056] Further, the washing and filtration medium for the first washing and filtration includes: a membrane package with a molecular weight cut-off of 300-1000 kD. Using a membrane package with a molecular weight cut-off of 300-1000 kD for the first washing and filtration can effectively remove sucrose and some impurities with relatively small molecular weights in the virus centrifugate. These small molecular impurities may include virus fragments, ovalbumin, etc. Their presence may affect the purity, safety, and immune effect of the vaccine. By removing these impurities, the quality of the vaccine can be significantly improved. The membrane package with a molecular weight cut-off of 300-1000 kD has a moderate cut-off molecular weight, which can not only effectively remove sucrose and small molecular impurities but also retain and recover most of the active ingredients. Using a membrane package with a molecular weight cut-off of 300-1000 kD for washing and filtration can simplify the purification process, reduce the number of operation steps, and thus improve the purification efficiency. At the same time, since the cut-off molecular weight of the membrane package is clear, the appropriate membrane package specifications can be selected according to needs to further optimize the purification process. By removing sucrose and small molecular impurities, the unstable factors during the storage and transportation of the vaccine can be reduced, thereby enhancing the stability of the vaccine. This is crucial for ensuring that the vaccine can still maintain its activity and immune effect after long-term storage.

[0057] Further, when performing the first ultrasonic treatment, the protein content in the influenza virus deglycosylated and purified solution is 2.0 - 3.0 mg / ml, including but not limited to any point value among 2.0 mg / ml, 2.2 mg / ml, 2.4 mg / ml, 2.6 mg / ml, 2.8 mg / ml or 3.0 mg / ml, or the range value between any two of them. Controlling the protein content in the influenza virus deglycosylated and purified solution within a certain range can significantly improve the lysis effect and efficiency, prevent insufficient lysis or over - lysis, thereby affecting the recovery rate of hemagglutinin and the immune effect. If the protein concentration is too high, the protein is more likely to aggregate, resulting in a shorter contact time between the lysis agent and the virus particles, and also reducing the amount of lysis agent effectively acting on the virus particles, prolonging the lysis time, leading to insufficient lysis, and even possibly the situation where some viruses cannot be effectively lysed. If the protein concentration is too low, the virus particles will be over - lysed, making the lysed virus particles smaller, and even possibly some active ingredients being lysed, reducing the recovery rate of hemagglutinin and the immune effect.

[0058] Further, the amplitude of the first ultrasonic treatment is 50% - 80%, including but not limited to any point value among 50%, 55%, 60%, 65%, 70%, 75% or 80%, or the range value between any two of them.

[0059] Further, the sample injection flow rate of the first ultrasonic treatment is ≤600 ml / min, including but not limited to any point value among 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min or 600 ml / min, or the range value between any two of them.

[0060] Further, the filtering medium for the filtration includes: a filter membrane with a pore size of 0.45 μm.

[0061] Further, the lysis agent for the lysis includes: at least one of Triton X - 100, polysorbate 80, Triton N101, ether, cetyltrimethylammonium bromide or sodium deoxycholate.

[0062] Further, when performing lysis, the concentration of the lysis agent is 0.5 vol% - 0.7 vol%, including but not limited to any point value among 0.5 vol%, 0.6 vol% or 0.7 vol%, or the range value between any two of them. This concentration range helps to ensure that the lysis agent can fully act on the virus particles, thereby effectively lysing the virus envelope and releasing the internal antigen components.

[0063] By precisely controlling the type and concentration of the lysing agent, the release amount and proportion of the effective antigen in the vaccine can be further optimized, thereby improving the immune effect of the vaccine. In addition, effective lysis also helps to reduce the impurity content in the vaccine, improving the purity and safety of the vaccine.

[0064] Further, the temperature of the lysis is 20-25°C, including but not limited to the point values of 20°C, 22°C, 24°C or 25°C or the range values between any two of them.

[0065] Further, the time of the lysis is 1.5-2.5 h, including but not limited to the point values of 1.5 h, 1.7 h, 1.9 h, 2.1 h, 2.3 h or 2.5 h or the range values between any two of them.

[0066] Further, the second washing and filtration includes: equal-volume washing and filtration.

[0067] Further, the lysing agent is removed by ultrafiltration using a membrane package with a molecular cut-off of 10-100 kD. Ultrafiltration using a membrane package with a molecular cut-off of 10-100 kD can efficiently remove the lysing agent in the influenza virus lysate. The removal of the lysing agent is one of the key steps in the vaccine purification process. By efficiently removing the lysing agent, the purity of the vaccine can be significantly improved, reducing potential impurities and contaminants and increasing the acceptance and trust of the vaccine. Using the ultrafiltration method to remove the lysing agent not only improves the removal efficiency but also simplifies the production process. Compared with traditional methods (such as chromatography, dialysis, etc.), the ultrafiltration method is more convenient and faster to operate. This helps to reduce production costs, improve production efficiency, and maintain the high quality of the vaccine.

[0068] Further, an inactivating agent is added to the purified influenza virus lysate with a protein content of 0.5-1.0 mg / ml (for example, it can be but not limited to the point values of 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml or 1.0 mg / ml or the range values between any two of them). This control range ensures that the inactivating agent can evenly contact the virus particles, thereby improving the inactivation efficiency. At the same time, an appropriate protein concentration helps to maintain the structural stability of the virus particles and reduce possible structural damage during the inactivation process. By precisely controlling the protein content before inactivation, it can be ensured that the inactivating agent can more effectively kill the virus while avoiding unnecessary damage to the effective antigen in the vaccine. This helps to improve the safety of the vaccine and reduce possible adverse reactions after vaccination.

[0069] Further, when performing the inactivation, the concentration of the inactivator is 190-200 μg / ml, including but not limited to any point value of 190 μg / ml, 195 μg / ml or 200 μg / ml or the range value between any two of them. This concentration range ensures that the inactivator can fully act on the virus particles, thereby effectively killing the virus. At the same time, an appropriate inactivator concentration also helps to reduce the residue of harmful substances that may be generated during the inactivation process.

[0070] Further, the inactivator includes: formaldehyde and / or β-propiolactone.

[0071] By precisely controlling the type and concentration of the inactivator, it can be ensured that the inactivation process causes the least damage to the effective antigen in the vaccine, while improving the stability and safety of the vaccine. In addition, the optimized inactivation conditions also help to reduce the impurity content and the risk of potential allergic reactions in the vaccine.

[0072] Further, the temperature of the inactivation is 2-8 °C, including but not limited to any point value of 2 °C, 4 °C, 6 °C or 8 °C or the range value between any two of them.

[0073] Further, the time of the inactivation is 115-125 h, including but not limited to any point value of 115 h, 118 h, 120 h, 123 h or 125 h or the range value between any two of them.

[0074] Further, the third washing and filtration includes: equal-volume washing and filtration.

[0075] Further, a membrane package with a cut-off molecular weight of 5-50 kD is used to remove the inactivator.

[0076] Currently, most methods use chromatography purification with a larger pore size membrane package to remove the inactivator from the inactivated influenza virus solution. However, the present invention uses a membrane package with a cut-off molecular weight of 10 kD for removal. The principle is that the membrane package has a specific pore size, and only substances with a molecular weight less than 10 kD can pass through the membrane package, while substances with a larger molecular weight are retained. Using this characteristic, the inactivator and other small molecule impurities are passed through the membrane package, and small molecule substances such as the inactivator can be removed through the membrane package, while macromolecular substances such as hemagglutinin are retained inside the membrane package.

[0077] This method of the present invention has obvious technical advantages. First, the hemagglutinin content of the recovered sample can be precisely controlled. An appropriate content of hemagglutinin can be retained as needed, which is beneficial to the preparation of the subsequent semi-finished product and ensures that the quality and performance of the product meet the requirements. Second, the yield of the target sample can be increased, and the purity of the sample is improved. Finally, this method is simple and efficient to operate. Compared with the chromatography purification method, the membrane package operation does not require complex equipment and cumbersome steps, reducing the operation time and labor costs. At the same time, the cost of the membrane package is relatively low, further reducing the production cost and improving the economic benefits of production.

[0078] Further, the amplitude of the second ultrasonic treatment is 50% - 80%, including but not limited to the point value of any one of 50%, 55%, 60%, 65%, 70%, 75% or 80% or the range value between any two of them.

[0079] Further, the sample injection flow rate of the second ultrasonic treatment ≤ 600 ml / min, including but not limited to the point value of any one of 50 ml / min, 100 ml / min, 150 ml / min, 200 ml / min, 250 ml / min, 300 ml / min, 350 ml / min, 400 ml / min, 450 ml / min, 500 ml / min, 550 ml / min or 600 ml / min or the range value between any two of them.

[0080] Further, the method for preparing the influenza virus concentrate includes the following steps:

[0081] (1) Preparation of influenza virus allantoic fluid harvest

[0082] Take the working seeds of H1N1 and BV influenza viruses, dilute them with 0.02 mol / L PBS buffer (pH 7.4), and then inoculate them into the allantoic cavity of 9 - 11-day-old chicken embryos, 0.20 ± 0.02 ml per embryo. Under the conditions of humidity 50% RH - 80% RH and temperature 33 - 35 °C, culture for 48 - 72 hours. For the qualified chicken embryos after embryo inspection, cold embryos for 10 - 22 hours at a temperature of 2 - 8 °C, and after centrifugal clarification with a disc centrifuge, it is the influenza virus allantoic fluid harvest.

[0083] (2) Preparation of influenza virus concentrate

[0084] The influenza virus allantoic fluid harvest is filtered and clarified through filters with pore sizes of 2.0 μm and 0.45 μm, then concentrated by a 750 kD hollow fiber ultrafiltration membrane, washed and filtered with 0.02 mol / L PBS buffer (pH 7.4) for no less than 5 times, and the concentration multiple is 10 - 15 times, which is the influenza virus concentrate.

[0085] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0086] The detailed information of the experimental materials or reagents involved in the embodiments of the present invention is as follows:

[0087] 1. Virulent strains: All influenza virus virulent strains are purchased from the National Institute for Biological Standards and Control (NIBSC), UK.

[0088] 2. Chicken embryos for virus passage and preparation are from SPF chicken flocks free of exogenous avian leukemia virus, exogenous avian adenovirus type I and type III, and healthy chicken embryos aged 9 - 11 days without deformities, with clear blood vessels and active.

[0089] 3. In the present invention, 9 - 11 - day - old chicken embryos hatched from Hy - Line White eggs meeting vaccine production requirements are selected, and influenza virus working seeds are used for large - scale amplification of the virus, and the virus solution is harvested.

[0090] 4. Preparation of 0.02 mol / L PBS buffer (pH 7.4): Weigh 5.80 g of Na2HPO4·12H2O, 0.59 g of NaH2PO4·2H2O and 8.50 g of NaCl, dilute to 1 L with injection water, and adjust the pH value of the solution to 7.4 with 2% sodium hydroxide solution.

[0091] Example 1

[0092] The preparation method of the high - dose influenza virus split vaccine stock solution provided in this example includes the following steps:

[0093] (1) Preparation of influenza virus allantoic fluid harvest

[0094] Take the working seeds of H1N1 and BV type influenza viruses, dilute them with 0.02 mol / L PBS buffer (pH 7.4), and then inoculate them into the allantoic cavity of 9 - 11 - day - old chicken embryos, 0.20 ± 0.02 ml per embryo. Under the conditions of 70% RH humidity and 34℃ temperature, culture for 65 hours. For the qualified chicken embryos after inspection, cool them at 5℃ for 16 hours, and then centrifuge and clarify them with a disc centrifuge to obtain the influenza virus allantoic fluid harvest.

[0095] (2) Preparation of influenza virus concentrate

[0096] The allantoic fluid of influenza virus is filtered and clarified through filters with pore sizes of 2.0 μm and 0.45 μm, concentrated by a 750 kD hollow fiber ultrafiltration membrane, washed and filtered with 0.02 mol / L PBS buffer (pH 7.4) for at least 5 times, and concentrated by 13 times to obtain the concentrated influenza virus solution.

[0097] (3) Preparation of the first centrifuged influenza virus solution

[0098] The concentrated influenza virus solution is purified by the first sucrose density gradient centrifugation. The concentration of the sucrose solution is 55%, the flow rate of the injection pump is 230 ml / min, the centrifugation speed is 35000 rpm. After centrifugation for 45 minutes, the pump flow rate is adjusted to 100 ml / min, and the sugar concentration is monitored online. The centrifuged solution in the sugar concentration range of 28% - 50% is collected, which is the first centrifuged influenza virus solution.

[0099] (4) Preparation of the second and third centrifuged influenza virus solutions

[0100] The first centrifuged influenza virus solution is diluted to 20 L with PBS buffer and subjected to the second sucrose density gradient centrifugation. The concentration of the sucrose solution is 55%, the flow rate of the injection pump is 230 ml / min, the centrifugation speed is 35000 rpm. After centrifugation for 45 minutes, the pump flow rate is adjusted to 100 ml / min, and the sugar concentration is monitored online. The centrifuged solutions in the sugar concentration ranges of 35% - 50% and 22% - 35% are collected respectively to obtain the second and third centrifuged influenza virus solutions.

[0101] (5) Preparation of the fourth centrifuged influenza virus solution

[0102] The third centrifuged influenza virus solution is diluted to 20 L with PBS buffer and subjected to the third sucrose density gradient centrifugation. The concentration of the sucrose solution is 55%, the flow rate of the injection pump is 230 ml / min, the centrifugation speed is 35000 rpm. After centrifugation for 45 minutes, the pump flow rate is adjusted to 100 ml / min, and the sugar concentration is monitored online. The centrifuged solution in the sugar concentration range of 35% - 50% is collected, which is the fourth centrifuged influenza virus solution.

[0103] (6) Preparation of the influenza virus desugared and purified solution

[0104] The second and fourth centrifuged influenza virus solutions are combined and diluted 4 times with PBS buffer; an equal volume washing and filtration is performed on the diluted centrifuged influenza virus solution using a membrane package with a cut-off molecular weight of 500 kD. The washing and filtration volume is not less than 10 times the volume of the diluted centrifuged influenza virus solution, and the recovered sample is the influenza virus desugared and purified solution.

[0105] (7) Preparation of the influenza virus lysate

[0106] Dilute the protein content of the influenza virus deglycosylated and purified solution to 2.5 mg / ml with PBS buffer. Subject the influenza virus deglycosylated and purified solution to continuous flow ultrasonic treatment. Set the ultrasonic amplitude to 65% and the ultrasonic injection flow rate to 600 ml / min. After ultrasonic treatment, filter it using a 0.45 μm pore size filter element. The solution after filtration is the influenza virus ultrasonic filtered and purified solution.

[0107] Add 10% TritonX-100 solution to the influenza virus ultrasonic filtered and purified solution to make its final concentration 0.6% (V / V). Shake and lyse at 23 °C for 2 hours. After lysis, it is the influenza virus lysate.

[0108] (8) Preparation of influenza virus lysed and purified solution

[0109] Use an ultrafiltration membrane package with a molecular weight cut-off of 50 kD to remove the lysing agent from the influenza virus lysate, and wash and filter with PBS buffer in an equal volume for no less than 5 times. It is the influenza virus lysed and purified solution.

[0110] (9) Preparation of influenza virus inactivated and purified solution

[0111] Dilute the protein content of the influenza virus lysed and purified solution to 0.8 mg / ml with PBS buffer; add 5% formaldehyde solution to the diluted influenza virus lysed and purified solution to make its final concentration 200 μg / ml, and inactivate it by magnetic stirring at 4 °C for a total of 120 hours. After inactivation, use a membrane package with a molecular weight cut-off of 10 kD to remove formaldehyde, and wash and filter with PBS buffer in an equal volume for no less than 2 times. It is the influenza virus inactivated and purified solution.

[0112] (10) Preparation of influenza virus monovalent stock solution

[0113] Subject the influenza virus inactivated and purified solution to 3 times of continuous flow ultrasonic treatment. Set the ultrasonic amplitude to 65% and the injection flow rate to 600 ml / min. After ultrasonic treatment, filter and sterilize it through a 0.2 μm filter. It is the influenza virus monovalent stock solution.

[0114] Example 2

[0115] The preparation method of the high-dose influenza virus lysed vaccine stock solution provided in this example includes the following steps:

[0116] (1) - (2) are the same as in Example 1.

[0117] (3) Preparation of the first influenza virus centrifuged solution

[0118] The influenza virus concentrate was purified by the first sucrose density gradient centrifugation. The concentration of the sucrose solution was 50%, the flow rate of the injection pump was 200 ml / min, the centrifugation speed was 34000 rpm. After centrifugation for 40 minutes, the pump flow rate was adjusted to 100 ml / min. The sugar concentration was monitored online, and the centrifugate in the sugar concentration range of 28% - 50% was collected, which was the first influenza virus centrifugate.

[0119] (4) Preparation of the second and third influenza virus centrifugates

[0120] The influenza virus centrifugate I was diluted to 20 L with PBS buffer and subjected to the second sucrose density gradient centrifugation. The concentration of the sucrose solution was 60%, the flow rate of the injection pump was 250 ml / min, the centrifugation speed was 36000 rpm. After centrifugation for 50 minutes, the pump flow rate was adjusted to 100 ml / min. The sugar concentration was monitored online, and the centrifugates in the sugar concentration ranges of 35% - 50% and 22% - 35% were collected respectively, obtaining the second and third influenza virus centrifugates.

[0121] (5) Preparation of the fourth influenza virus centrifugate

[0122] The third influenza virus centrifugate was diluted to 20 L with PBS buffer and subjected to the third sucrose density gradient centrifugation. The concentration of the sucrose solution was 50%, the flow rate of the injection pump was 200 ml / min, the centrifugation speed was 34000 rpm. After centrifugation for 40 minutes, the pump flow rate was adjusted to 100 ml / min. The sugar concentration was monitored online, and the centrifugate in the sugar concentration range of 35% - 50% was collected, which was the fourth influenza virus centrifugate.

[0123] (6) Same as Example 1.

[0124] (7) Preparation of the influenza virus lysate

[0125] The protein content of the influenza virus desugared and purified solution was diluted to 3.0 mg / ml with PBS buffer. The influenza virus desugared and purified solution was subjected to continuous flow ultrasonic treatment. The ultrasonic amplitude was set at 65%, the ultrasonic injection flow rate was 600 ml / min. After ultrasonic treatment, it was filtered using a 0.45 μm pore size filter element, and the filtered solution was the influenza virus ultrasonic filtered and purified solution.

[0126] A 10% TritonX - 100 solution was added to the influenza virus ultrasonic filtered and purified solution to make its final concentration 0.5% (V / V), and it was shaken and lysed at 23°C for 2 hours. After the lysis was completed, it was the influenza virus lysate.

[0127] (8) Same as Example 1.

[0128] (9) Preparation of the inactivated and purified influenza virus solution

[0129] Dilute the protein content of the influenza virus lysate and purified solution to 0.5 mg / ml with PBS buffer; add 5% formaldehyde solution to the diluted influenza virus lysate and purified solution to make its final concentration 200 μg / ml, inactivate it by magnetic stirring at 4°C for a total of 120 hours. After inactivation, use a membrane package with a cut-off molecular weight of 10 kD to remove formaldehyde, and wash and filter with PBS buffer in an equal volume for no less than 2 times, which is the inactivated and purified influenza virus solution.

[0130] (10) The same as Example 1.

[0131] Example 3

[0132] The preparation method of the high-dose influenza virus lysate vaccine stock solution provided in this example includes the following steps:

[0133] (1)-(2) The same as Example 1.

[0134] (3) Preparation of the first influenza virus centrifugate

[0135] Perform the first sucrose density gradient centrifugation and purification on the influenza virus concentrate. The concentration of the sucrose solution is 60%, the flow rate of the injection pump is 250 ml / min, the centrifugation speed is 36000 rpm. After centrifugation for 50 minutes, adjust the pump flow rate to 100 ml / min, monitor the sugar concentration online, and collect the centrifugate in the sugar concentration range of 28%-50%, which is the first influenza virus centrifugate.

[0136] (4) Preparation of the second influenza virus centrifugate and the third influenza virus centrifugate

[0137] Dilute the influenza virus centrifugate I to 20 L with PBS buffer, and perform the second sucrose density gradient centrifugation. The concentration of the sucrose solution is 50%, the flow rate of the injection pump is 200 ml / min, the centrifugation speed is 34000 rpm. After centrifugation for 40 minutes, adjust the pump flow rate to 100 ml / min, monitor the sugar concentration online, and collect the centrifugate in the sugar concentration ranges of 35%-50% and 22%-35% respectively, to obtain the second influenza virus centrifugate and the third influenza virus centrifugate.

[0138] (5) Preparation of the fourth influenza virus centrifugate

[0139] Dilute the third influenza virus centrifugate to 20 L with PBS buffer, and perform the third sucrose density gradient centrifugation. The concentration of the sucrose solution is 60%, the flow rate of the injection pump is 250 ml / min, the centrifugation speed is 36000 rpm. After centrifugation for 50 minutes, adjust the pump flow rate to 100 ml / min, monitor the sugar concentration online, and collect the centrifugate in the sugar concentration range of 35%-50%, which is the fourth influenza virus centrifugate.

[0140] (6) The same as Example 1.

[0141] (7) Preparation of Influenza Virus Lysate

[0142] Dilute the protein content of the influenza virus deglycosylated and purified solution to 2.0 mg / ml with PBS buffer. Continuously flow the influenza virus deglycosylated and purified solution through ultrasonic treatment, set the ultrasonic amplitude to 65%, the ultrasonic injection flow rate to 600 ml / min. After ultrasonic treatment, filter it with a 0.45 μm pore size filter element, and the filtered solution is the ultrasonic filtered and purified influenza virus solution.

[0143] Add 10% TritonX-100 solution to the ultrasonic filtered and purified influenza virus solution to make its final concentration 0.7% (V / V), and shake and lyse at 23 °C for 2 hours. After the lysis is completed, it is the influenza virus lysate.

[0144] (8) The same as Example 1.

[0145] (9) Preparation of Inactivated and Purified Influenza Virus Solution

[0146] Dilute the protein content of the influenza virus lysed and purified solution to 1.0 mg / ml with PBS buffer; add 5% formaldehyde solution to the diluted influenza virus lysed and purified solution to make its final concentration 190 μg / ml, and inactivate it by magnetic stirring at 4 °C for a total of 120 hours. After inactivation, use a 10 kD molecular weight cut-off membrane package to remove formaldehyde, and wash and filter with PBS buffer in an equal volume for no less than 2 times, which is the inactivated and purified influenza virus solution.

[0147] (10) The same as Example 1.

[0148] Comparative Example 1

[0149] The preparation method of the high-dose influenza virus lysate vaccine stock solution provided in this comparative example includes the following steps:

[0150] (1) The same as step (1) of Example 1.

[0151] (2) The same as step (2) of Example 1.

[0152] (3) Preparation of Influenza Virus Centrifuged Solution

[0153] The influenza virus concentrate is purified by sucrose density gradient centrifugation. The concentration of the sucrose solution is 55%, the flow rate of the injection pump is 230 ml / min, the centrifugation speed is 35000 rpm. After centrifugation for 45 minutes, the pump flow rate is adjusted to 100 ml / min, and the sugar concentration is monitored online. Collect the influenza virus centrifuged solution in the sugar concentration range of 35% - 50%, and store the collected virus centrifuged solution at 4 °C.

[0154] (4) Preparation of Influenza Virus Deglycosylated and Purified Solution

[0155] Ultrafiltration was performed using a membrane package with a molecular weight cut-off of 500 kD to remove sucrose. The sample was washed and filtered with an equal volume of 0.02 mol / L PBS buffer (pH 7.4) for no less than 10 times. After the washing and filtering were completed, the recovered sample was the defucosylated and purified influenza virus solution.

[0156] (5) Preparation of inactivated influenza virus solution

[0157] The protein content of the defucosylated and purified influenza virus solution was diluted to 1950 μg / ml with 0.02 mol / L PBS buffer (pH 7.4). The diluted defucosylated and purified influenza virus solution was subjected to continuous-flow ultrasonic treatment. The ultrasonic amplitude was set at 65%, and the inlet flow rate was 600 ml / min. Immediately after ultrasonic treatment, filtration was performed through a filter with a pore size of 0.45 μm, and the resulting solution was the ultrasonically filtered and purified influenza virus solution.

[0158] A 5% formaldehyde solution was added to the ultrasonically filtered and purified influenza virus solution to a final concentration of 200 μg / ml, and it was placed in a magnetic stirrer at 4°C for inactivation for 144 h. After inactivation, it was the inactivated influenza virus solution.

[0159] (6) Preparation of inactivated and purified influenza virus solution

[0160] The inactivated influenza virus solution was purified by chromatography on Sepharose 6FF gel. The sample loading volume did not exceed 10% of the column volume, and the chromatography flow rate was 35 cm / h ± 5 cm / h. 0.02 mol / L PBS buffer (pH 7.4) was used as the eluent, and the absorption peak was collected through a 280 nm ultraviolet detector, controlling the protein content within the range of 700 - 1200 μg / ml.

[0161] (7) Preparation of lysed and purified influenza virus solution

[0162] A 10% TritonX-100 solution was added to the inactivated and purified influenza virus solution to a final concentration of 0.5% - 0.7% (V / V), and it was placed in a magnetic stirrer at 23°C for lysis for 2 h. After lysis, ultrafiltration was performed using a membrane package with a molecular weight cut-off of 50 kD to remove the lysing agent, and it was washed and filtered 15 times with an equal volume of 0.02 mol / L PBS buffer (pH 7.4), which was the lysed and purified influenza virus solution.

[0163] (8) Preparation of monovalent influenza virus stock solution

[0164] The lysed and purified influenza virus solution was subjected to 3 consecutive continuous-flow ultrasonic treatments. The ultrasonic amplitude was set at 65%, and the ultrasonic sample injection flow rate was 600 ml / min. The sample after ultrasonic treatment was filtered through a 0.2 μm filter for sterilization, which was the monovalent influenza virus stock solution.

[0165] Comparative Example 2

[0166] The difference between this comparative example and Example 1 lies only in that in step (3), the centrifugate in the sugar content range of 45% - 55% is collected as the first influenza virus centrifugate.

[0167] Comparative Example 3

[0168] The difference between this comparative example and Example 1 lies only in that in step (4), the centrifugate in the sugar content range of 45% - 55% is collected as the second influenza virus centrifugate, and the centrifugate in the sugar content range of 35% - 55% is collected as the third influenza virus centrifugate.

[0169] Comparative Example 4

[0170] The difference between this comparative example and Example 1 lies only in that in step (5), the centrifugate with a sugar content range of 50% - 60% is collected, which is the fourth influenza virus centrifugate.

[0171] Experimental Example

[0172] The influenza virus monovalent stock solutions obtained in each example and comparative example were subjected to component detection, and the detection results are shown in Table 1.

[0173] Table 1

[0174]

[0175]

[0176] According to the comparative analysis of the data in Table 1, it can be seen that the influenza virus monovalent stock solution of Example 1 prepared by the process of the present invention has significant advantages in key quality indicators. The experimental data show that the protein hemagglutination ratio and ovalbumin content of each type of product in Example 1 are significantly lower than those of other comparative examples.

[0177] It should be noted that except for Comparative Example 1 which uses the traditional single - centrifugation process, the remaining experimental groups all applied an innovative process system of three - stage gradient density centrifugation, virus lysis treatment before inactivation, and removal of inactivator with a 10KD ultrafiltration membrane package. Particularly noteworthy is that in the stock solutions of each type prepared by this process system, the difference in ovalbumin content between different types is significantly reduced, which indicates a successful breakthrough in the problem of quality differences between types in the traditional process.

[0178] In addition, the protein hemagglutination ratios of these groups are also lower than that of Comparative Example 1 using the single - centrifugation process. These results strongly prove the effectiveness and necessity of the preparation process of the present invention.

[0179] The influenza virus desugared and purified solutions obtained in each example and comparative example were subjected to component detection, and the detection results are shown in Table 2.

[0180] Table 2

[0181]

[0182]

[0183] As can be seen from Table 2, the concentrated solution was purified by gradient density centrifugation and deglycosylation purification process to obtain the deglycosylated and purified influenza virus solution. The specific test results are detailed in Table 2.

[0184] To more clearly evaluate the process effect, the deglycosylated and purified influenza virus solution was compared and analyzed with the concentrated influenza virus solution. The results showed that the hemagglutinin recovery rates of each type in Example 1 were excellent and were significantly higher than those in other examples. At the same time, the protein hemagglutination ratios of each type in Example 1 were also lower than those in other examples.

[0185] It should be noted that except for Comparative Example 1, the remaining examples and comparative examples all adopted the three - step gradient density centrifugation process. The hemagglutinin recovery rates of each type in Comparative Example 1 were only higher than those in Comparative Example 2 and were at a disadvantage in other comparisons; moreover, the protein hemagglutination ratios of each type were significantly higher than those in other examples.

[0186] In summary, the three - step gradient density centrifugation process has significant benefits for improving the hemagglutinin recovery rate and reducing the protein hemagglutination ratio, which strongly proves the effectiveness and necessity of the preparation process of the present invention.

[0187] Although the present invention has been illustrated and described with specific examples, it should be realized that the above - mentioned examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them; those of ordinary skill in the art should understand that: without departing from the spirit and scope of the present invention, the technical solutions recorded in the foregoing examples can be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the examples of the present invention; therefore, this means that all such replacements and modifications that fall within the scope of the present invention are included in the appended claims.

Claims

1. A method for preparing a high-dose influenza virus split vaccine stock solution, characterized in that: The following steps are involved: (a) subjecting the influenza virus concentrate to a first sucrose density gradient centrifugation, collecting the centrifuge with a sugar content of 28% to 50%, and obtaining a first influenza virus centrifuge; subjecting the first influenza virus centrifuge to a second sucrose density gradient centrifugation, collecting the centrifuge with a sugar content of 35% to 50% and 22% to 35%, respectively, and obtaining a second influenza virus centrifuge and a third influenza virus centrifuge; (b) subjecting the third influenza virus centrifuge to a third sucrose density gradient centrifugation, collecting the centrifuge with a sugar content of 35% to 50%, and obtaining a fourth influenza virus centrifuge; (c) mixing and diluting the second influenza virus centrifuge liquid and the fourth influenza virus centrifuge liquid, and then performing a first diafiltration to obtain an influenza virus desugared purified liquid; (d) subjecting the influenza virus desugared purified solution to a first ultrasonic treatment, filtering, and then lysing to obtain an influenza virus lysate; and subjecting the influenza virus lysate to a second diafiltration to remove the lysing agent in the influenza virus lysate to obtain an influenza virus lysate purified solution; (e) adding an inactivator to the influenza virus lysate purified solution to inactivate the solution, thereby obtaining an influenza virus inactivated solution; and performing a third filtration to remove the inactivator from the influenza virus inactivated solution, thereby obtaining an influenza virus inactivated purified solution; The influenza virus inactivated purified solution is subjected to a second ultrasonic treatment and sterile filtration to obtain an influenza virus monovalent stock solution.

2. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The concentration of the sucrose solution in the first sucrose density gradient centrifugation is 50wt% to 60wt%; (2) the injection flow rate of the first sucrose density gradient centrifugation is 200-250 ml / min; (3) the speed of the first sucrose density gradient centrifugation is 34000-36000 rpm; (4) The time of the first sucrose density gradient centrifugation is 40 to 50 minutes.

3. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The concentration of the sucrose solution in the second sucrose density gradient centrifugation is 50wt% to 60wt%; (2) the injection flow rate of the second sucrose density gradient centrifugation is 200-250 ml / min; (3) the speed of the second sucrose density gradient centrifugation is 34000-36000 rpm; (4) The time of the second sucrose density gradient centrifugation is 40 to 50 minutes.

4. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: Includes at least one of the following technical features: (1) The concentration of the sucrose solution in the third sucrose density gradient centrifugation is 50wt% to 60wt%; (2) the injection flow rate of the third sucrose density gradient centrifugation is 200-250 ml / min; (3) the speed of the third sucrose density gradient centrifugation is 34000-36000 rpm; (4) The time of the third sucrose density gradient centrifugation is 40 to 50 minutes.

5. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: The diafiltration medium of the first diafiltration comprises a membrane package with a molecular cutoff of 300 to 1000 kD.

6. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: Using a membrane package with a molecular cutoff of 10 to 100 kD to ultrafilter and remove the lysing agent; And / or, use a membrane package with a molecular weight cutoff of 5 to 50 kD to remove the inactivator.

7. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: During the first ultrasonic treatment, the protein content of the influenza virus desugared purified solution is 2.0-3.0 mg / ml.

8. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: An inactivating agent is added to the influenza virus lysis purification solution having a protein content of 0.5 to 1.0 mg / ml.

9. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: The cleavage agent comprises: at least one of TritonX-100, polysorbate 80, Triton N101, ether, hexadecyltrimethylammonium bromide or sodium deoxycholate; And / or, during the lysis, the concentration of the lysis agent is 0.5 vol% to 0.7 vol%.

10. The method for preparing a high-dose influenza virus split vaccine stock solution according to claim 1, characterized in that: When performing the inactivation, the concentration of the inactivator is 190-200 μg / ml; And / or, the inactivator includes: formaldehyde and / or β-propiolactone.

Images