CN117442714A - Novel varicella attenuated live vaccine and preparation method thereof - Google Patents

Novel varicella attenuated live vaccine and preparation method thereof Download PDF

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CN117442714A
CN117442714A CN202311800745.1A CN202311800745A CN117442714A CN 117442714 A CN117442714 A CN 117442714A CN 202311800745 A CN202311800745 A CN 202311800745A CN 117442714 A CN117442714 A CN 117442714A
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solution
virus
live vaccine
attenuated live
varicella
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梁智杰
杨旭
李占春
蒋佳丽
陈小伟
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Boaovax Biotechnology Co ltd
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Abstract

The invention discloses a novel varicella attenuated live vaccine and a preparation method thereof, and belongs to the technical field of varicella attenuated live vaccines. The preparation method comprises the following steps: performing continuous cell subculture by adopting a cell growth solution containing 8-10vol% of bovine serum; then changing the cell growth liquid into a virus culture liquid, inoculating and culturing the strain, discarding the virus culture liquid, washing, and adding a virus maintenance liquid for continuous culture; when the lesion area reaches more than 70%, removing the virus maintenance solution, rinsing, adding phosphate buffer solution for ultrasonic treatment; collecting cytopathic matters obtained by ultrasonic treatment, centrifuging, and adding a freeze-drying stabilizer for re-suspension to obtain virus harvest. The invention can simply and efficiently obtain the varicella attenuated live vaccine with stable and excellent quality, and the obtained vaccine has low bovine serum albumin and other impurity content.

Description

Novel varicella attenuated live vaccine and preparation method thereof
Technical Field
The invention relates to the technical field of varicella attenuated live vaccines.
Background
Varicella is a common infectious disease, is high in children, and is strong in infectivity. Varicella vaccine is an attenuated live virus vaccine, namely varicella attenuated live vaccine, which is prepared by varicella virus passaging strain, is the only means for preventing varicella infection, and the varicella vaccine can be used for preventing varicella and complications caused by varicella-zoster.
The preparation method of the common varicella attenuated live vaccine in the prior art is mainly a direct infection method, such as culturing human embryo lung diploid cells through bovine serum MEM growth solution with the volume percentage content of more than 12%, then directly inoculating Oka strain virus into the cultured cells obtained through the growth solution, and then obtaining virus harvest through the processes of removing the more complex cell culture solution, digesting the human embryo lung diploid cells and the like.
The above methods generally have the following drawbacks: the virus harvesting process is tedious, time-consuming and labor-consuming, so that the quality of virus inactivation stock solution in the preparation is obviously reduced, and meanwhile, the problem of high bovine serum albumin residue in the obtained varicella attenuated live vaccine is easy to occur.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a novel preparation method of a varicella attenuated live vaccine and the varicella attenuated live vaccine, the preparation method can obtain the varicella attenuated live vaccine with stable and excellent quality simply and efficiently through cell culture solution and virus culture solution with low bovine serum content and ultrasonic treatment, and simultaneously, the preparation method has the advantages of less toxic seed consumption, less raw material consumption and low bovine serum albumin and other impurity content in the obtained vaccine.
The technical scheme of the invention is as follows:
a method for preparing a novel varicella attenuated live vaccine, comprising:
(1) Continuously subculturing MRC-5 strain human embryo lung diploid cells by adopting a cell growth solution at 37+/-1 ℃;
(2) After the cell culture is amplified to 38 generations, changing the cell growth liquid into a virus culture liquid, inoculating the Oka strain in the virus culture liquid according to the concentration of 0.01-0.005 M.O.I, and culturing for 2 days at 35+/-1 ℃ after the inoculation is finished;
(3) After confirming that the cytopathic area reaches 10-30%, discarding the virus culture solution, adding PBS solution for rinsing 2-3 times according to 200-300 ml/layer cell factory, adding virus maintenance solution according to the liquid amount of 150-250 ml/layer cell factory, and continuously culturing at 35+/-1 ℃;
(4) Removing the virus maintenance solution after the cytopathic area reaches more than 70%, adding a phosphate buffer solution for rinsing for 1 time, adding the phosphate buffer solution according to the liquid amount of 80-120 ml/layer cell factory after removing the washing solution, carrying out ultrasonic treatment, collecting the obtained infected cells after ultrasonic treatment, carrying out centrifugal treatment, removing the supernatant after centrifugal treatment, adding a freeze-drying stabilizer for resuspension sediment according to the liquid amount of 5-15% of the total volume before centrifugation, mixing uniformly, and obtaining a virus harvest, and freezing and storing the virus harvest below-60 ℃;
(5) Re-thawing the prepared frozen virus harvest, adding a freeze-drying stabilizer for dilution and combination, and filtering to obtain a stock solution;
(6) Introducing the stock solution into a semi-finished product bottle by using a sealing pipeline to obtain a semi-finished product;
(7) Subpackaging and freeze-drying the obtained semi-finished product to obtain the novel varicella attenuated live vaccine;
wherein,
the cell growth liquid consists of 8-10% by volume of bovine serum, 2-3% by volume of 3%L-glutamine solution and the balance E' MEM solution, and the pH value is regulated to 7.2+/-0.2;
the 3%L-glutamine solution contains the following components: 0.03g/ml L-glutamine, 0.0068g/ml NaCl, 0.0004gKCl/ml, caCl 0.0002g/ml 2 ·2H 2 MgCl of O, 0.00017g/ml 2 ·6H 2 O, 0.000158g/ml NaH 2 PO 4 ·H 2 O and 0.0011g/ml glucose, and the solvent is water for injection;
the E ' MEM solution is an aqueous solution of E ' MEM dry powder, wherein the content of the E ' MEM dry powder is 0.0094g/ml;
the virus culture solution consists of 2-5% of bovine serum by volume percentage, 2-3% of 3%L-glutamine solution by volume percentage and the balance E' MEM solution, and the pH value is regulated to 7.6+/-0.2;
the PBS solution contains the following components: 0.008g/ml NaCl, 0.0004g/ml KCl, 0.00006g/ml KH 2 PO4, 0.000132 g/ml Na 2 HPO 4 ·12H 2 O and 0.001g/ml glucose, and the solvent is water for injection;
the virus maintenance solution consists of 0.5-1.5% of human serum albumin, 2-3% of 3%L-glutamine solution and the balance E' MEM solution by volume percentage, and the pH value is regulated to 7.6+/-0.2.
Preferably, the diluting and combining in step (5) includes: and adding the solution of the freeze-drying stabilizer into the re-melted virus harvest, and diluting until the virus titer is 5.0-5.3 lg PFU/ml.
Preferably, in the step (5), a filter with a pore size of 90-150 μm is used for the filtration.
Preferably, the phosphate buffer contains the following components: 2.126 ×10 -3 g/ml NaCl, 0.344X 10 - 3 g/ml NaH 2 PO 4 ·H 2 O and 5X 10 -3 g/ml Na 2 HPO 4 ·12H 2 O, the solvent is water for injection.
Preferably, the ultrasonic treatment uses an extracellular ultrasonic device, the ultrasonic power is 80-90%, the ultrasonic frequency is 20000Hz, the air source pressure is 0.4-0.5 Mpa, the ultrasonic time is 1.2-1.5 s, and the environmental temperature is 18-26 ℃.
Preferably, the centrifugal force of the centrifugal treatment is 10000-15200 Xg, and the centrifugal time is 15-20 min.
Preferably, the temperature of the centrifugal treatment is 2-8 ℃.
Preferably, the pH is adjusted by 5.6% NaHCO 3 Solution implementation, 5.6% NaHCO 3 The solution is NaHCO 3 In which NaHCO 3 Is 0.056g/ml.
Preferably, the freeze-drying stabilizer comprises the following components: sucrose, trehalose, sodium glutamate, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and human serum albumin.
The invention has the following beneficial effects:
(1) According to the invention, cell culture can be performed through E' MEM growth solution of 8-10vol% of bovine serum, and the high-quality attenuated live vaccine is obtained, the content of the used bovine serum is low, and the residual bovine serum albumin in the finished product is lower than that in the prior art.
(2) The invention can directly obtain the virus harvest by ultrasonic treatment without the process of cell digestion by EDTA solution, solves the problems of complicated operation, time and labor waste and reduced quality of virus inactivation stock solution in the prior art, and reduces the foreign impurities in the finished product.
(3) The inoculation of the poisoning seeds is only 0.01-0.005 M.O.I, so that the dosage of the poisoning seeds can be reduced, the raw materials can be saved, the efficiency can be improved, and the cost can be reduced.
(4) The varicella attenuated live vaccine meeting the titer requirement and having no inter-batch difference can be prepared by adding the freeze-drying stabilizer for dilution, so that the quality stability of the obtained vaccine is improved.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all, of the embodiments of the present invention. All other embodiments, based on the embodiments of the invention, which are obtained by a person skilled in the art without any inventive effort, are within the scope of the invention.
The following examples were prepared using the following solutions or reagents:
e' MEM solution: 9.40g of E' -MEM dry powder is weighed and dissolved in a small amount of water for injection, after the dry powder is fully dissolved, the volume is fixed to 1000ml, and the dry powder is sterilized, filtered, packaged and stored at the temperature of 2-8 ℃ for later use.
Cell growth liquid: consists of 8-10% by volume of bovine serum, 2-3% by volume of 3%L-glutamine solution and the balance E' MEM solution, and passes through 5.6% NaHCO 3 The pH of the solution was adjusted to 7.2.+ -. 0.2.
3%L-glutamine solution: weighing 30.00g of L-glutamine powder, 6.80g of NaCl, 0.40g of KCl and 0.20g of CaCl 2 ·2H 2 O、0.17g MgCl 2 ·6H 2 O、0.158g NaH 2 PO 4 ·H 2 O and 1.10g glucose are dissolved in a small amount of water for injection, after the O and the glucose are fully dissolved, the volume is fixed to 1000ml, the sterilization and the filtration are carried out, and then the split charging is carried out, and the storage is carried out at the temperature of 2-8 ℃ for standby.
5.6%NaHCO 3 Solution: weighing 56.00g NaHCO 3 And dissolving the powder in a small amount of water for injection, after the powder is fully dissolved, fixing the volume to 1000ml, sterilizing, filtering, sub-packaging, and preserving at 2-8 ℃ for later use.
Virus culture solution: consists of 2% by volume of bovine serum, 2% by volume of 3%L-glutamine solution and the balance E' MEM solution, and is prepared by mixing 5.6% NaHCO 3 The pH value of the solution is regulated to 7.6+/-0.2.
PBS solution: weighing 8.00g NaCl, 0.40g KCl and 0.06g KH 2 PO4、0.132g Na 2 HPO 4 ·12H 2 O and 1.00g glucose are dissolved in a small amount of water for injection, after the O and the glucose are fully dissolved, the volume is fixed to 1000ml, and the water for injection is sterilized, filtered, split-packed and stored at the temperature of 2-8 ℃ for standby.
Virus maintenance solution: consists of 0.5% by volume of human serum albumin, 2% by volume of 3%L-glutamine solution and the balance E' MEM solution, and passes through 5.6% NaHCO 3 The pH value of the solution is regulated to 7.6+/-0.2.
Phosphate buffer: 2.126g NaCl, 0.344g NaH were weighed out 2 PO 4 ·H 2 O and 5g Na 2 HPO 4 ·12H 2 O is dissolved inAnd (3) adding a small amount of water for injection, after the water for injection is fully dissolved, fixing the volume to 1000ml, sterilizing, filtering, sub-packaging, and preserving at 2-8 ℃ for later use.
Freeze-drying stabilizer: the formulation was as shown in Table 1:
table 1 freeze-dried stabilizer formulation
Example 1
A varicella attenuated live vaccine is prepared by the steps of:
(1) Continuously subculturing MRC-5 strain human embryo lung diploid cells by adopting a cell growth solution, wherein the culture temperature is 37+/-1 ℃, and culturing and amplifying to 38 generations; the pH value of the cell growth liquid is 7.0-7.4, and the cell growth liquid is E' MEM growth liquid containing 10vol% of bovine serum;
(2) After the cells are cultured for 3 days to form a compact monolayer, changing a cell growth solution into a virus culture solution, and inoculating a virus seed, wherein the virus seed adopts an Oka strain varicella virus, inoculating the cells according to 0.01-0.005 M.O.I, and culturing for 2 days at 35+/-1 ℃ after the inoculation is finished;
(3) After the cells inoculated with the virus are cultured for 2 days at the temperature of 35+/-1 ℃, the cytopathic area reaches 10-30%, the virus culture solution is discarded, PBS solution is added according to the liquid amount of 300 ml/layer of cell factory for rinsing for 2-3 times, after the rinsing is finished, virus maintenance solution is added according to the liquid amount of 200 ml/layer of cell factory, and the cells are placed at the temperature of 35+/-1 ℃ for continuous culture;
(4) After the cytopathic area reaches more than 70%, removing virus maintenance solution, adding phosphate buffer solution for rinsing 1 time according to the liquid amount of a cell factory of 100 ml/layer, removing washing solution, adding phosphate buffer solution according to the liquid amount of the cell factory of 100 ml/layer, performing ultrasonic treatment at the environment temperature of 18-26 ℃ by using ultrasonic equipment outside the cell factory, wherein the ultrasonic time is 1.2-1.5 s, the ultrasonic power is 80-90%, the ultrasonic frequency is 20000Hz, the air source pressure is 0.40-0.50 Mpa, collecting infected cells after ultrasonic treatment, merging and uniformly mixing, subpackaging into a centrifugal cup for centrifugal treatment at the temperature of 2-8 ℃, the centrifugal force is 14400g, the centrifugal time is 20min, removing supernatant after the centrifugal treatment, adding freeze-drying stabilizer according to the liquid amount of 10% of the total volume before the centrifugal treatment, re-suspending sediment, and obtaining virus harvest after uniform mixing; freezing at below-60deg.C;
(5) Taking out frozen virus harvest prepared from the same cell batch from a refrigerator below minus 60 ℃, re-melting at room temperature, adding a freeze-drying stabilizer, diluting and merging according to virus titer of more than 5.1LgPFU/ml, and filtering by using a filter with aperture of 150 mu m to obtain stock solution;
(6) Introducing the stock solution into a semi-finished product bottle through a sealed pipeline, and uniformly stirring to obtain a semi-finished product;
(7) And subpackaging and freeze-drying the obtained semi-finished product to obtain the varicella attenuated live vaccine.
Example 2
A varicella attenuated live vaccine is prepared by the steps of:
(1) Continuously subculturing MRC-5 strain human embryo lung diploid cells by adopting a cell growth solution, wherein the culture temperature is 37+/-1 ℃, and culturing and amplifying to 38 generations; the pH value of the cell growth liquid is 7.0-7.4, and the cell growth liquid is E' MEM growth liquid containing 8vol% of bovine serum;
(2) After the cells are cultured for 3 days to form a compact monolayer, changing a cell growth solution into a virus culture solution, and inoculating a virus seed, wherein the virus seed adopts an Oka strain varicella virus, inoculating the cells according to 0.005 M.O.I, and culturing for 2 days at 35+/-1 ℃ after the inoculation is finished;
(3) After the cells inoculated with the virus are cultured for 2 days at 35+/-1 ℃, the cytopathic area reaches 10-30%, the virus culture solution is discarded, PBS solution is added according to the liquid amount of 300 ml/layer of cell factory for rinsing 3 times, after the rinsing is finished, virus maintenance solution is added according to the liquid amount of 200 ml/layer of cell factory, and the cells are placed at 35+/-1 ℃ for continuous culture;
(4) After the cytopathic area reaches more than 70%, removing virus maintenance solution, adding phosphate buffer solution for rinsing 1 time according to the liquid amount of a cell factory of 100 ml/layer, removing washing solution, adding phosphate buffer solution according to the liquid amount of the cell factory of 100 ml/layer, performing ultrasonic treatment by using an external ultrasonic device of the cell factory at the ambient temperature (18-26 ℃) for 1.2-1.5 seconds, performing ultrasonic power for 80-90%, performing ultrasonic frequency for 20000Hz and air source pressure for 0.40-0.50 mpa, collecting infected cells after ultrasonic treatment, merging and uniformly mixing, subpackaging into a centrifugal cup for centrifugal treatment at the temperature of 2-8 ℃, performing centrifugal force for 15200g and centrifugal time for 20min, removing supernatant after centrifugal treatment, adding freeze-drying stabilizer heavy suspension sediment according to the liquid amount of 15% of the total volume before centrifugal treatment, and obtaining virus harvest after uniform mixing; freezing at below-60deg.C;
(5) Taking out frozen virus harvest prepared from the same cell batch from a refrigerator below minus 60 ℃, completing re-melting at room temperature, adding a freeze-drying stabilizer, diluting and merging according to the virus titer of more than 5.1LgPFU/ml, and filtering by using a filter with the aperture of 120 mu m to obtain a stock solution;
(6) And (3) introducing the stock solution into a semi-finished product bottle, uniformly stirring, sub-packaging, and freeze-drying to obtain the varicella attenuated live vaccine.
Example 3
A varicella attenuated live vaccine is prepared by the steps of:
(1) Continuously subculturing MRC-5 strain human embryo lung diploid cells by adopting a cell growth solution, wherein the culture temperature is 37+/-1 ℃, and culturing and amplifying to 38 generations; the pH value of the cell growth liquid is 7.0-7.4, and the cell growth liquid is E' MEM growth liquid containing 10vol% of bovine serum;
(2) After the cells are cultured for 3 days, the cells form a compact monolayer, the cell growth liquid is replaced by a virus culture liquid, and virus seed inoculation is carried out, wherein the virus seed adopts the varicella virus of the Oka strain, the cells are inoculated according to 0.005 M.O.I, and after the inoculation is completed, the cells are cultured for 2 days at 35+/-1 ℃;
(3) After the cells inoculated with the virus are cultured for 2 days at the temperature of 35+/-1 ℃, the cytopathic area reaches 10-30%, the virus culture solution is discarded, PBS solution is added according to the liquid amount of 300 ml/layer of cell factory for rinsing 2 times, after the rinsing is finished, virus maintenance solution is added according to the liquid amount of 250 ml/layer of cell factory, and the cells are placed at the temperature of 35+/-1 ℃ for continuous culture;
(4) Removing virus maintenance solution after the cytopathic area reaches more than 70%, adding phosphate buffer solution for rinsing 1 time according to the liquid amount of a cell factory of 100 ml/layer, removing washing solution, adding phosphate buffer solution according to the liquid amount of the cell factory of 100 ml/layer, performing ultrasonic treatment by using an extracellular ultrasonic device at the ambient temperature (18-26 ℃) for 1.2-1.5 seconds, performing ultrasonic power of 80-90%, ultrasonic frequency of 20000Hz and air source pressure of 0.45Mpa, collecting infected cells after ultrasonic treatment, merging and uniformly mixing, subpackaging into a centrifugal cup for centrifugal treatment at the temperature of 2-8 ℃, performing centrifugal force of 14400g and centrifugal time of 20min, removing supernatant after centrifugal treatment, adding a freeze-drying stabilizer for re-suspending sediment according to the liquid amount of 10% of the total volume before centrifugal treatment, and obtaining a virus harvest after mixing uniformly; freezing at below-60deg.C;
(5) Taking out frozen virus harvest prepared from the same cell batch from a refrigerator below minus 60 ℃, completing re-melting at room temperature, adding a freeze-drying stabilizer, diluting and merging according to the virus titer of more than 5.1LgPFU/ml, and filtering by using a filter with the aperture of 90 mu m to obtain a stock solution;
(6) Introducing the stock solution into a semi-finished product bottle through a sealed pipeline, and uniformly stirring to obtain a semi-finished product;
(7) And subpackaging and freeze-drying the obtained semi-finished product to obtain the varicella attenuated live vaccine.
The residual amounts of bovine serum albumin in the viral harvest, stock and final products prepared in examples 1, 2 and 3 were measured, and the results are shown in table 2 below:
TABLE 2 detection results of virus titer and impurity content of examples 1 to 3
As can be seen from the above detection results, the residual amounts of bovine serum albumin in the varicella attenuated live vaccine finished products obtained in examples 1 to 3 are all low, and the residual amounts in example 2 are the lowest. The invention can efficiently obtain excellent products with low bovine serum albumin residual quantity under the production mode of a large-scale cell factory.
The above embodiment is a preferred embodiment of the present invention, and the scope of the present invention is not limited to the above embodiment. All technical schemes belonging to the concept of the invention belong to the protection scope of the invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (10)

1. A method for preparing a novel varicella attenuated live vaccine, which is characterized by comprising the following steps:
(1) Continuously subculturing MRC-5 strain human embryo lung diploid cells by adopting a cell growth solution at 37+/-1 ℃;
(2) After the cell culture is amplified to 38 generations, changing the cell growth liquid into a virus culture liquid, inoculating the Oka strain in the virus culture liquid according to the concentration of 0.01-0.005 M.O.I, and culturing for 2 days at 35+/-1 ℃ after the inoculation is finished;
(3) After confirming that the cytopathic area reaches 10-30%, discarding the virus culture solution, adding PBS solution for rinsing 2-3 times according to 200-300 ml/layer cell factory, adding virus maintenance solution according to the liquid amount of 150-250 ml/layer cell factory, and continuously culturing at 35+/-1 ℃;
(4) Removing the virus maintenance solution after the cytopathic area reaches more than 70%, adding a phosphate buffer solution for rinsing for 1 time, adding the phosphate buffer solution according to the liquid amount of 80-120 ml/layer cell factory after removing the washing solution, carrying out ultrasonic treatment, collecting the obtained infected cells after ultrasonic treatment, carrying out centrifugal treatment, removing the supernatant after centrifugal treatment, adding a freeze-drying stabilizer for resuspension sediment according to the liquid amount of 5-15% of the total volume before centrifugation, mixing uniformly, and obtaining a virus harvest, and freezing and storing the virus harvest below-60 ℃;
(5) Re-thawing frozen virus harvest at room temperature, adding freeze-drying stabilizer, diluting and combining, and filtering to obtain stock solution;
(6) Introducing the stock solution into a semi-finished product bottle by using a sealing pipeline to obtain a semi-finished product;
(7) Subpackaging and freeze-drying the obtained semi-finished product to obtain the novel varicella attenuated live vaccine;
wherein,
the cell growth liquid consists of 8-10% by volume of bovine serum, 2-3% by volume of 3%L-glutamine solution and the balance E' MEM solution, and the pH value is regulated to 7.2+/-0.2;
the 3%L-glutamine solution contains the following components: 0.03g/ml L-glutamine, 0.0068g/ml NaCl, 0.0004g/ml KCl, 0.0002g/ml CaCl 2 ·2H 2 MgCl of O, 0.00017g/ml 2 ·6H 2 O, 0.000158g/ml NaH 2 PO 4 ·H 2 O and 0.0011g/ml glucose, and the solvent is water for injection;
the E ' MEM solution is an aqueous solution of E ' MEM dry powder, wherein the content of the E ' MEM dry powder is 0.0094g/ml;
the virus culture solution consists of 2-5% of bovine serum by volume percentage, 2-3% of 3%L-glutamine solution by volume percentage and the balance E' MEM solution, and the pH value is regulated to 7.6+/-0.2;
the PBS solution contains the following components: 0.008g/ml NaCl, 0.0004g/ml KCl, 0.00006g/ml KH 2 PO4, 0.000132 g/ml Na 2 HPO 4 ·12H 2 O and 0.001g/ml glucose, and the solvent is water for injection;
the virus maintenance solution consists of 0.5-1.5% of human serum albumin, 2-3% of 3%L-glutamine solution and the balance E' MEM solution by volume percentage, and the pH value is regulated to 7.6+/-0.2.
2. The method for preparing a novel varicella live attenuated vaccine according to claim 1, wherein the dilution combining in the step (5) comprises: and adding the solution of the freeze-drying stabilizer into the re-melted virus harvest, and diluting until the virus titer is 5.0-5.3 lg PFU/ml.
3. The method for preparing a novel varicella attenuated live vaccine according to claim 1, wherein the filtration in the step (5) uses a filter having a pore size of 90 to 150 μm.
4. Novel varicella reduction according to claim 1The preparation method of the live vaccine is characterized in that the phosphate buffer solution comprises the following components: 2.126 ×10 -3 g/ml NaCl, 0.344X 10 -3 g/ml NaH 2 PO 4 ·H 2 O and 5X 10 -3 g/ml Na 2 HPO 4 ·12H 2 O, the solvent is water for injection.
5. The method for preparing the novel varicella attenuated live vaccine according to claim 1, wherein the ultrasonic treatment is performed by using an extracellular ultrasonic device, the ultrasonic power is 80-90%, the ultrasonic frequency is 20000Hz, the air source pressure is 0.4-0.5 mpa, the ultrasonic time is 1.2-1.5 s, and the environmental temperature is 18-26 ℃.
6. The method for preparing the novel varicella attenuated live vaccine according to claim 1, wherein the centrifugal force of the centrifugal treatment is 10000-15200 Xg, and the centrifugal time is 15-20 min.
7. The method for preparing a novel varicella attenuated live vaccine according to claim 1, wherein the temperature of the centrifugation treatment is 2-8 ℃.
8. The method for preparing a novel attenuated varicella live vaccine according to claim 1, wherein the pH value is adjusted by 5.6% NaHCO 3 Solution implementation, 5.6% NaHCO 3 The solution is NaHCO 3 In which NaHCO 3 Is 0.056g/ml.
9. The method for preparing a novel varicella attenuated live vaccine according to claim 1, wherein the freeze-drying stabilizer comprises the following components: sucrose, trehalose, sodium glutamate, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and human serum albumin.
10. The novel varicella attenuated live vaccine prepared by the method for preparing a novel varicella attenuated live vaccine according to any one of claims 1 to 9.
CN202311800745.1A 2023-12-26 2023-12-26 Novel varicella attenuated live vaccine and preparation method thereof Pending CN117442714A (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000256A (en) * 1975-04-30 1976-12-28 Merck & Co., Inc. Varicella vaccine and process for its preparation
US6210683B1 (en) * 1997-09-05 2001-04-03 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
CN1593657A (en) * 2004-07-09 2005-03-16 长春天坛生物制药有限公司 Production method of freeze dried chickenpox attenuated live vaccine and products therefrom
CN101161286A (en) * 2007-09-06 2008-04-16 上海荣盛生物技术有限公司 Varicella attenuation live vaccine
CN104258404A (en) * 2014-09-11 2015-01-07 长春长生生物科技股份有限公司 Freeze-dried vaccine protective agent, freeze-dried varicella attenuated live vaccine and preparation methods of freeze-dried vaccine protective agent and freeze-dried varicella attenuated live vaccine
CN108753736A (en) * 2018-05-04 2018-11-06 上海荣盛生物药业有限公司 Produce the method and its application of varicella virus stoste
CN114292805A (en) * 2022-01-10 2022-04-08 中国原子能科学研究院 Method for fully extracting total protein of adherent cells

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000256A (en) * 1975-04-30 1976-12-28 Merck & Co., Inc. Varicella vaccine and process for its preparation
US6210683B1 (en) * 1997-09-05 2001-04-03 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
CN1593657A (en) * 2004-07-09 2005-03-16 长春天坛生物制药有限公司 Production method of freeze dried chickenpox attenuated live vaccine and products therefrom
CN101161286A (en) * 2007-09-06 2008-04-16 上海荣盛生物技术有限公司 Varicella attenuation live vaccine
CN104258404A (en) * 2014-09-11 2015-01-07 长春长生生物科技股份有限公司 Freeze-dried vaccine protective agent, freeze-dried varicella attenuated live vaccine and preparation methods of freeze-dried vaccine protective agent and freeze-dried varicella attenuated live vaccine
CN108753736A (en) * 2018-05-04 2018-11-06 上海荣盛生物药业有限公司 Produce the method and its application of varicella virus stoste
CN114292805A (en) * 2022-01-10 2022-04-08 中国原子能科学研究院 Method for fully extracting total protein of adherent cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
李生军: "带状疱疹疫苗制备工艺的研究", 中国硕士电子期刊工程科技Ⅰ辑, 15 March 2020 (2020-03-15) *
陈晓彤等: "贴壁细胞超声脱壁技术研究", 集成技术, vol. 11, no. 5, 30 September 2022 (2022-09-30), pages 1 - 11 *

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