Method for producing vaccine by virtue of culturing animal cells

A technology of animal cells and cultured cells, which is applied in the field of bioengineering and can solve problems such as low degree of automation, large differences in product batches, unstable quality of cells and vaccines, etc.

Inactive Publication Date: 2011-10-05

HANGZHOU AMPROTEIN BIOENG +1

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AI-Extracted Technical Summary

Problems solved by technology

[0003] At present, the production of vaccines is mainly carried out through the spinning bottle process, but this method has a low degree of automation and high labor intensity, and because of the uncontrollable environment of the spinning bottle cell culture, the quality of the cells and vaccines is unstable, and the product batches vary greatly; On the other hand, t...

Abstract

The invention belongs to the field of vaccine preparation, and provides a method for producing a vaccine by virtue of culturing animal cells in a flake carrier. The method comprises the following steps: culturing vaccine stromal cells on the flake carrier; inoculating virus based on a certain concentration; continuously culturing cells and virus under a bioreactor control condition to obtain a virus solution; repeatedly freezing and thawing the flake virus and infection cells; and mixing all collected solutions to synthesize a virus suspension so as to prepare the vaccine. In the method, a flake carrier culturing technology is applied to producing the vaccine by virtue of culturing the animal cells in the bioreactor, the flake carrier provides a three-dimensional space and a larger specific surface area for cell growth and virus breeding, the cell density of the cells is higher, and the bioreactor controls culture conditions and other parameters to provide the best producing environment for cell growth and virus breeding, so that the virus titer is greatly reduced, and the vaccine yield and quality are more stable.

Application Domain

Vertebrate cellsArtificial cell constructs +2

Technology Topic

Virus suspensionTiter +10

Examples

Experimental program(5)

Example

[0027] Example 1 (Preparation of PRRS vaccine by culturing Marc-145 cells in a 10L bioreactor)

[0028] Porcine reproductive and respiratory syndrome virus: CH-1R strain.

[0029] After the sheet-like carrier is sterilized by gamma ray 24kGy irradiation, it is first soaked in PBS, then rinsed with PBS, and then soaked in culture solution overnight for use.

[0030] Inoculate 1 x 10 on the sheet carrier in the bioreactor 7 Marc-145 cells were cultured and grown for 5 to 7 days under the conditions of DO controlled at 30-60%, pH value maintained at 7.0-7.2, and temperature of 37 °C, during which glucose digestion was detected, and the medium was changed according to the sugar consumption. , so that the daily sugar consumption is maintained above 1g/L, and with the increase of cell density, the daily sugar consumption gradually increases until it reaches more than 2.5g/L.

[0031] The culture medium was drained, washed twice with sterile PBS, and inoculated with porcine reproductive and respiratory syndrome virus at a multiplicity of infection (MOI) of 0.001-0.01. Among them, porcine reproductive and respiratory syndrome virus PRRSV, whether European type or American type, virulent strain or attenuated strain, generally has adaptability and sensitivity to Marc-145 cells or other susceptible cells, so the method of the present invention Applicable to all PRRSV strains.

[0032] Continue to culture the susceptible cell Marc-145, adopt fed-feed culture mode, maintain pH at 7.2-7.4, DO at 30-60%, temperature at 37°C, and nutrient conditions required for virus propagation. After the virus infects susceptible cells for a period of time (10-20 hours), the virus maintenance solution or glucose concentrate can be added to the bioreactor by means of fed-batch culture to maintain proper nutrients, and the cells are harvested after 30-40 hours. virus fluid. The harvested supernatant virus solution was placed at -20°C, and the sheet-like vector and cells were repeatedly freeze-thawed at -20°C for 2 to 3 times, and then mixed with the supernatant virus solution to prepare a virus suspension, which was prepared by freeze-drying after passing the test. Get the PRRS vaccine.

[0033] Determination of virus titer TCID50 was performed according to conventional methods. The virus titer of the virus liquid collected in the harvest stage was increased by 1-2 TCID50/ml compared with that of the spinner bottle culture process (that is, the virus titer was 10% higher than that of the spinner bottle culture process). 1-2 times), the volume of each harvest is 10 times that of the spinning bottle, and the total virus yield is 100 times that of the spinning bottle process.

[0034] After testing, the results of vaccine quality control meet the requirements of the 2005 edition of the Chinese Pharmacopoeia and the 2000 edition of the Chinese Biological Products Regulations.

[0035] Replacing the N-type sheet-shaped carrier in this embodiment with polygonal shapes such as M, W, Z, star, pentagon, square, or rhombus can also achieve the same technical effect, which will not be repeated here.

Example

[0036] Example 2 (10L bioreactor cultivates MDCK cells to prepare avian influenza vaccine)

[0037] After the sheet-like carrier is sterilized by gamma ray 24kGy irradiation, it is first soaked in PBS, then rinsed with PBS, and then soaked in culture solution overnight for use.

[0038] Inoculate 0.5 x 10 on the sheet carrier in the bioreactor 7 For MDCK cells, DO was controlled at 30-60%, pH was maintained at 7.2-7.4, and temperature was 37°C. Glucose digestion was detected, and liquid exchange or rehydration treatment was carried out according to the sugar consumption, so that the sugar content was maintained above 1 g/L, and the cell density reached the maximum value in 5 to 7 days.

[0039] The culture medium was emptied, washed twice with sterile PBS, and inoculated with H5N1 virus at a multiplicity of infection (MOI) of 0.01-0.1. The methods of the present invention are applicable to all strains that are generally adaptive and sensitive to MDCK cells or other susceptible cells.

[0040] Continue to culture the susceptible cell MDCK, adopt the fed-batch culture method, maintain pH at 7.3-7.5, DO at 30-60%, temperature at 37°C, and nutrient conditions required for virus reproduction. After the virus infects the susceptible cells for a period of time, such as 10-20 hours, the virus maintenance solution or glucose concentrate can be added to the bioreactor by means of fed-batch culture, and the virus solution can be harvested after 3-4 days. The harvested supernatant virus solution was placed at -20°C, the sheet-like carrier and cells were repeatedly frozen and thawed at -20°C for 3 times, and then mixed with the supernatant virus solution to make a virus suspension, filtered, inactivated, and emulsified. Preparation of avian influenza vaccine.

[0041] The determination of viral hemagglutination titer was performed according to conventional methods. Compared with the rolling bottle process, the viral HA of the virus liquid collected in the harvest stage is increased by 4-8 HA, that is, the virus titer is 2 times that of the rolling bottle culture process. 2-3 Compared with the chicken embryo process, the viral HA of the virus liquid is increased by 1-2 HA, that is, the virus titer is 2 times that of the chicken embryo culture process. 1-2 times.

[0042] After testing, the results of vaccine quality control meet the requirements of the 2005 edition of the Chinese Pharmacopoeia and the 2000 edition of the Chinese Biological Products Regulations.

[0043] Replacing the N-type sheet-shaped carrier in this embodiment with polygonal shapes such as M, W, Z, star, pentagon, square, or rhombus can also achieve the same technical effect, which will not be repeated here.

Example

[0044] Example 3 (10L bioreactor culture ST cells to prepare swine fever vaccine)

[0045] After the sheet-like carrier is sterilized by gamma ray 24kGy irradiation, it is first soaked in PBS, then rinsed with PBS, and then soaked in culture solution overnight for use.

[0046] Inoculate 2 x 10 on the sheet carrier in the bioreactor 7 For each ST cell, DO was controlled at 30-60%, pH was maintained at 7.0-7.2, and temperature was 37°C. Glucose digestion was detected, and the medium was changed every 2 to 3 days according to the sugar consumption, so that the sugar content was maintained above 1 g/L, and the maximum cell density was reached in 5 to 7 days.

[0047] All the culture medium was drained, the sheet-like carrier and cells were washed twice with sterile PBS, and were inoculated into ST cells at a concentration of 2-3% spleen poison. The methods of the present invention are applicable to all strains that are generally adaptive and sensitive to ST cells or other susceptible cells.

[0048] Continue culturing the ST cells by means of fed-batch culture and batch harvesting, maintaining pH at 7.2-7.4, DO at 30-60%, temperature at 37°C, and nutrient conditions required for virus propagation. After the virus infects the susceptible cells, the sugar consumption is detected every 12 hours. The virus maintenance solution or glucose concentrate can be added to the bioreactor by means of fed-batch culture. The virus solution is harvested on the 5th day after inoculation. Harvest once a day. The harvested supernatant virus liquid was freeze-dried to prepare a swine fever vaccine.

[0049] The determination of titer was carried out according to the stereotype thermal reaction test method. The virus titer of the virus liquid collected at the harvest stage is compared with that of the rolling bottle culture process. The best case of the rolling bottle process can reach ≥ 100,000 rabbits per milliliter of virus infection, and the swine fever vaccine produced by sheet carrier culture can reach 100,000 swine fever vaccine per milliliter. The infection rate of rabbits containing virus ≥ 500,000 is increased by more than 5 times.

[0050] After testing, the results of vaccine quality control meet the requirements of the 2005 edition of the Chinese Pharmacopoeia and the 2000 edition of the Chinese Biological Products Regulations.

[0051] Replacing the N-type sheet-shaped carrier in this embodiment with polygonal shapes such as M, W, Z, star, pentagon, square, or rhombus can also achieve the same technical effect, which will not be repeated here.

PUM

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