CN102660510B - A kind of method utilizing bio-reactor to produce transmissible gastro-enteritis virus - Google Patents

Abstract

The invention provides a method for producing porcine transmissible gastroenteritis virus by using a bioreactor, comprising the following steps: 1) preparing monolayer passage cells; 2) preparing seed poison for porcine transmissible gastroenteritis virus production; 3) preparing porcine transmissible gastroenteritis virus Step 1) The monolayer passaged cells prepared are prepared into a cell suspension, inoculated into a bioreactor, and the passaged cells are adsorbed and cultivated on the microcarriers in the bioreactor; 4) when the passaged cells grow to 80% of the microcarriers- 90%, the empty ball rate is less than 5%, the full ball rate is greater than 80%, and when the cell count reaches 3-5×10 ⁶ cells/mL or more, inoculate the seed virus prepared in step 2) with an inoculation amount of 2-5%, Carry out the adsorption culture of the virus; 4) when more than 80% of the passaged cells on the microcarriers are diseased, the virus liquid is harvested. The method of the invention can solve the problems of low production efficiency, unstable product quality and low virus titer, can increase the virus unit culture titer by 5-10 times, comprehensively improve the quality and yield of vaccines, and improve the safety of vaccines.

Description

A method for producing porcine transmissible gastroenteritis virus using a bioreactor

technical field

The invention relates to a method for producing a virus, in particular to a method for producing porcine transmissible gastroenteritis virus, and more specifically to a method for producing porcine transmissible gastroenteritis virus by utilizing a bioreactor.

Background technique

Porcine transmissible gastroenteritis (Porcine Transmissible Gastroenteritis, TGE) is caused by porcine transmissible gastroenteritis virus (Porcine Transmissible Gastroenteritis virus, TGEV) of Coronaviridae (Coronaviridae) and Coronavirus (Coronavirus), characterized by pig vomiting, diarrhea, and dehydration An infectious disease that has a high lethality rate for newborn piglets, usually 100%. Although pigs of different ages are susceptible to the virus, pigs older than 5 weeks have a low mortality rate after infection. After Doyle reported that the disease first occurred in the United States in 1945, many countries in the world have reported porcine transmissible gastroenteritis one after another. After the disease first occurred in Guangdong, my country in 1956, it also occurred in most provinces of the country, which brought great harm to the pig industry. Serious harm.

At present, the production of porcine transmissible gastroenteritis vaccine in China only relies on the cell spinner bottle culture method. The traditional process is labor-intensive, time-consuming, low in efficiency, and high in production cost; it is easy to be polluted by the environment; there are large differences between different production batches or between different bottles of the same production batch; it is difficult to expand production; it is easy to be contaminated by bacteria or other viruses The hidden dangers of vaccine quality caused by this problem involve biosafety and public health issues.

Contents of the invention

In order to solve the above problems, the present invention provides a method for producing porcine transmissible gastroenteritis virus using a bioreactor.

The method provided by the invention comprises the steps of:

1) Prepare monolayer passage cells;

2) preparing porcine transmissible gastroenteritis virus seed virus for production;

3) Digest the monolayer passaged cells prepared in step 1), suspend them with cell growth medium, prepare a cell suspension with a cell density of 1-3×10 ⁵ cells/mL, inoculate them into a bioreactor, and make the passaged cells Cells are adsorbed and cultured on microcarriers in a bioreactor;

4) When the passaged cells grow to 80%-90% of the microcarrier, the empty ball rate is lower than 5%, the full ball rate is greater than 80%, and the cell count reaches 3-5×10 ⁶ cells/mL or more, use 2-5 % inoculum amount inoculates the seed virus prepared in step 2), and carries out the adsorption culture of the virus;

4) When more than 80% of the passaged cells on the microcarriers are diseased, the virus liquid is harvested.

Wherein, the method also includes adding sterile cell growth liquid according to 50-70% of the total volume of the bioreactor before the cell suspension in step 3) is inoculated into the bioreactor, and every liter of sterile cell growth liquid Add microcarriers at a concentration of 4-10g/L, start the bioreactor, and stir at low speed for 20-50 minutes.

Wherein, the method also includes the steps of cleaning and sterilizing the microcarriers before adding the microcarriers to the cell growth solution, followed by soaking the microcarriers with PBS for more than 3 hours; washing the microcarriers with PBS for 3-5 times; Soak microcarriers in PBS and steam sterilize at 121°C for 20-50min.

Wherein, step 2) the preparation of seed virus for porcine transmissible gastroenteritis virus production specifically includes the following steps:

Inoculate porcine transmissible gastroenteritis virus seed virus into single-layer passage cells at a ratio of 2-5% with virus culture liquid for cultivation, and harvest the virus liquid when more than 80% of the cells are diseased; then use this virus liquid as the seed virus, Carry out cell adaptive continuous passage and rejuvenation on the cells, and the virus TCID ₅₀ and sterility test are carried out for each passage product, and the rejuvenation is stopped when the virus TCID ₅₀ is stable and reaches more than 10 ⁷ TCID ₅₀ /mL, and used as a seed virus for production. Wherein, the virus culture solution can be a conventional virus culture solution in the field, and the preferred formula is: the mass fraction is 98%-99% MEM solution, 1%-2% bovine serum, and the final concentration is 100-200IU/ mL of antibiotics, pH 6.8-7.5.

Wherein, the monolayer passaged cells in step 3) are digested with EDTA-trypsin digestion solution, and the EDTA-trypsin digestion solution is containing 0.05%-0.25% trypsin and 0.02% EDTA in mass volume fraction Hank'S liquid.

Wherein, the density of passaged cells in the cell suspension described in step 3) is 1-3×10 ⁵ cells/mL.

Wherein, the formula of the cell growth liquid described in step 3) is: the mass fraction is 95%-98% MEM liquid, 2%-5% bovine serum, the antibiotic of final concentration is 100-200IU/mL, and pH value is 6.8 -7.5.

Wherein, the passaged cells may be cells routinely used for virus culture in the art, preferably ST cells or vero cells, most preferably ST cells.

In the present invention, the parameters set in the bioreactor are preferably: cultivation temperature 35-38°C, pH value 6.8-7.5, dissolved oxygen 50%-70%, stirring speed 50-80rpm.

Wherein, the porcine transmissible gastroenteritis virus can be any conventional strain of porcine transmissible gastroenteritis virus, preferably a floating feather vaccine strain, h-5 vaccine strain or Sinovirus strain.

Wherein, the microcarriers can be any conventional microcarriers in the art, such as macroporous gelatin microcarriers, polystyrene microcarriers, PHEMA microcarriers, chitin microcarriers, polyurethane foam microcarriers, alginate gel microcarriers, etc. Gel microcarriers, magnetic microcarriers, Cytodex1, Cytodex2, Cytodex3, Cytopore and/or Cytoline, preferably Cytodex1, Cytodex2, Cytodex3, Cytodex and/or Cytoline.

Compared with the prior art, the present invention has the following beneficial effects:

(1) Using bioreactor microcarrier cell culture technology instead of spinner bottle cell culture technology to manufacture porcine transmissible gastroenteritis virus vaccine can solve the problems of low production efficiency, unstable product quality and low virus titer. Through production technology and The change of the production process can increase the virus unit culture titer by 5-10 times, comprehensively improve the quality and yield of the vaccine, and improve the safety of the vaccine. Taking vero cells as an example, domestic advanced bioreactor production can reach 10 ⁷ /mL, while spinner bottle production is generally 10 ⁶ /mL is already the limit.

(2) The present invention can greatly reduce the production cost, and the production cycle is short, and each production cycle only needs 5-6 days, which is significantly shorter than the 6-7 days of the existing spinner bottle cell culture method.

(3) Low pollution: 1 can = 100-2000 spinner bottles, the probability is that the fewer the operation links, the lower the probability of pollution.

(4) The production process is controllable: high-precision culture, real-time monitoring of dissolved oxygen, gas production, PH, cell status, etc., comprehensive parameter setting of the culture curve, directly ensuring the status of the carrier cells; large batches, small differences between batches; saving Space, personnel (up to 1:75); once the molding process will be very stable step by step.

Description of drawings

Fig. 1 is a flow chart of the method for producing porcine transmissible gastroenteritis virus using a bioreactor according to the present invention.

detailed description

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

Example 1

(1) Choose a bioreactor as the means of cultivation: 7L Swiss Lambda bioreactor.

(2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex2.

(3) Cleaning and sterilization methods of microcarriers: 1) Weigh 10 g/L of Cytodex2 microcarriers and soak in 2LPBS solution for 3 hours. 2) Wash 3 times with 2LPBS each time. 3) Add 2LPBS solution to soak the microcarriers, and steam sterilize at 121° C. for 30 minutes.

(4) Select ST cells as cells for making seedlings.

(5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged by EDTA-trypsin (Hank'S solution containing 0.25% trypsin and 0.02% EDTA), and then prepared with 95% MEM solution, 5% calf serum, 200IU /mL of penicillin sodium and streptomycin sulfate, the pH value of the cell growth medium was adjusted to 7.2, and the culture temperature was 37°C. When a good monolayer is formed, it is used to inoculate in a bioreactor for microcarrier suspension culture.

(6) Propagation of cytotoxic species: with virus culture fluid (containing 2% MEM liquid of calf serum, penicillin sodium and streptomycin sulfate that the final concentration is 200IU/mL, porcine transmissible gastroenteritis virus Hua strain The seed virus was inoculated into the above-mentioned well-grown cell monolayer according to the ratio of 2% by volume, and continued to cultivate at a temperature of 35°C. Harvest the virus liquid when more than 80% of the cells were diseased; measure the TCID ₅₀ of the virus, and wait until the virus titer is stable and Rejuvenation was stopped when it reached above 10 ⁷ TCID ₅₀ /mL, and the virus was used as a seed virus for production.

(7) Microcarrier suspension culture of ST in a bioreactor: take well-grown ST cells on a square bottle, wash them once with PBS with a pH value of 7.2, add the aforementioned EDTA-trypsin digestion solution for digestion, and wait for the cell layer to start If the whole piece falls off, add the above-mentioned cell growth solution, blow and suspend to make a cell suspension, and inoculate the reactor at a cell density of 2×10 ⁵ /mL. The bioreactor was set with the following parameters: temperature 37° C., pH value 7.2, stirring speed 60 rpm, dissolved oxygen content 50%, and the reactor was automatically controlled for cultivation.

(8) Propagation of seedling venom: on the third day after cultivation, observe that the cells on the microcarriers are basically overgrown (over 95%), and after the cell counting result reaches over 4×10 ⁶ /mL, inoculate at 2% by volume Virus. The culture parameters such as temperature 34°C, pH value 6.8, stirring speed 70rpm, dissolved oxygen content 30% were set, and the reactor was automatically controlled for cultivation. After inoculation, the microcarriers in the reactor were taken at regular intervals, the cell pathological changes were observed with a microscope, and the TCID ₅₀ of the samples was detected. To observe that the cells on the microcarriers are basically all shedding (about 36h), and the DO value is obviously rising, stop the reactor stirring, and after the microcarriers all sink to the bottom of the reactor, harvest the supernatant and the microcarriers respectively.

Processing of harvested virus liquid: after 3 times of repeated freezing and thawing, the harvested supernatant was filtered to remove cell debris and then frozen at -20°C for later use.

Example 2

(1) Choose a bioreactor as the means of cultivation: 7L Swiss Lambda bioreactor.

(2) Select the microcarrier as the carrier for cell attachment and growth: microcarrier Cytodex3.

(3) Cleaning and sterilization methods of microcarriers: 1) Weigh Cytodex3 microcarriers at 6 g/L and soak in 2 LPBS solution for 3 hours. 2) Wash 3 times with 2LPBS each time. 3) Add 2LPBS solution to soak the microcarriers, and steam sterilize at 121° C. for 30 minutes.

(4) Select vero cells as cells for seedling production.

(5) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged by EDTA-trypsin (Hank'S solution containing 0.25% trypsin and 0.02% EDTA), and then prepared with 96% MEM solution, 4% calf serum, 200IU /mL of penicillin sodium and streptomycin sulfate, the pH value of the cell growth medium was adjusted to 7.2, and the culture temperature was 37°C. When a good monolayer is formed, it is used to inoculate in a bioreactor for microcarrier suspension culture.

(6) Propagation of cytotoxic species: pig infectious gastrointestinal Inoculate the well-growing monolayer of the above-mentioned cells with 5% volume ratio of the seed virus of the Hua strain of phlogiston virus, and continue culturing at a temperature of 35°C. Harvest the virus liquid when more than 80% of the cells are diseased; measure the TCID ₅₀ of the virus, and stop rejuvenation when the virus titer is stable and reaches more than 10 ⁷ TCID ₅₀ /mL, and use the virus as a seed virus for production.

(7) Microcarrier suspension culture of vero cells in a bioreactor: take the well-grown vero cells on the square bottle, wash them once with PBS with a pH value of 7.2, add the aforementioned EDTA-trypsin digestion solution for digestion, and wait for the cell layer When the entire sheet begins to fall off, the cell growth solution is added, and the cell suspension is formed by blowing, and the cell count density is 1×10 ⁵ /mL and inoculated into the reactor. The bioreactor was set with the following parameters: temperature 37° C., pH value 7.2, stirring speed 60 rpm, and dissolved oxygen content 50% to carry out automatic reactor control cultivation.

(8) Propagation of seedling venom: after the third day of cultivation, observe that the cells on the microcarrier are basically overgrown (over 95%), and after the cell counting result reaches more than 5×10 ⁶ /mL, proceed with a volume ratio of 2%. Viral inoculation. Set the culture parameters such as temperature at 38°C, pH value at 7.5, stirring speed at 50 rpm, and dissolved oxygen content at 50% to carry out reactor automatic control culture. The virus fluid was harvested two days later. Processing of harvested virus liquid: after 3 times of repeated freezing and thawing, the harvested supernatant was filtered to remove cell debris and then frozen at -20°C for later use.

Comparative Example 1 Spinner bottle cultivation of porcine transmissible gastroenteritis virus

(1) Choose spinner bottle as the means of culture: 3L cell culture spinner bottle, ZP-01-16 spinner bottle machine.

(2) Select vero cells as cells for seedling production.

(3) Passaging and culturing of cells for seedling preparation: the above-mentioned cells were digested and passaged by EDTA-trypsin (Hank'S solution containing 0.25% trypsin and 0.02% EDTA), and then prepared with 95% MEM solution, 5% calf serum, 200IU /mL of penicillin sodium and streptomycin sulfate, the pH value of the cell growth medium was adjusted to 7.2, and the culture temperature was 37°C. When a good monolayer is formed, it is used for inoculation in roller bottle culture.

(4) Propagation of cytotoxic species: with virus culture fluid (containing the MEM liquid of serum 1%, the penicillin sodium and the streptomycin sulfate that the final concentration is 200IU, the pH value is adjusted to 7.3), the porcine transmissible gastroenteritis virus The seed virus of the Sinovirus strain was inoculated into the well-grown monolayer of the above-mentioned cells at a volume ratio of 5%, and the culture was continued at a temperature of 35°C. Harvest the virus liquid when more than 80% of the cells are diseased; measure the TCID ₅₀ of the virus, and stop rejuvenation when the virus titer is stable and reaches more than 10 ⁷ TCID ₅₀ /mL, and use the virus as a seed virus for production.

(5) Cultivation of vero cells in spinner flasks: Take the well-grown vero cells on the square bottle and wash them with PBS with a pH value of 7.2, add the EDTA-trypsin digestion solution to digest, and wait until the cell layer begins to appear as a whole After falling off, add the above-mentioned cell growth solution, blow suspension to make a cell suspension, and inoculate a 3000mL spinner bottle with a cell count density of 3×10 ⁵ /mL, with a culture volume of 300mL in the spinner bottle, 37°C, 20rpm/h, and culture until the cells Grow in a single layer.

(6) Propagation of seedling venom: observe on the third day after culture, the cells on the spinner bottle wall are basically overgrown (more than 95%), and the virus inoculation is carried out after the cell count result reaches more than 5×10 ⁶ /mL. The virus was inoculated at an inoculum volume ratio of 2%, and the virus supernatant was harvested 4 days later. Processing of harvested virus liquid: after 3 times of repeated freezing and thawing, the harvested supernatant was filtered to remove cell debris and then frozen at -20°C for later use.

Test example 1 Semi-finished product inspection

1. Determination of the poisonous value of the harvested virus liquid: the cell virus liquid harvested above is mixed and then sampled, and the poisonous value is determined.

The results showed that the virus content of the virus produced by the bioreactor in Examples 1 and 2 was ≥10 ⁷ TCID ₅₀ /mL, while the virus content of the comparative example 1 produced by spinner bottle culture was ≤10 ⁶ TCID ₅₀ /mL.

2. Inspection of semi-finished products after inactivation

Inactivation: Add formaldehyde solution to the virus liquids of Examples 1-2 and Comparative Example 1 at 0.2% of the total amount, shake for 2 minutes, and inactivate at 37° C. for 24 hours, stirring and mixing regularly during this period.

Sterility test: According to the appendix 301 page of the "Quality Standards for Veterinary Biological Products of the People's Republic of China", the viruses of the examples of the present invention and the comparative examples grew aseptically.

Inactivation test: After inactivation, take a sample from the virus inactivation solution by aseptic operation, inoculate a single layer of healthy ST cells at a ratio of 1%, and culture it at 37°C for 3 days. Pathological changes are qualified, and embodiment of the present invention and comparative example all have no pathological changes, so product is qualified.

3. Comparison of the immune effects of virus cultured in bioreactors and spinner bottles

Take 200 μL of virus with a titer of 1×10 ⁶ /mL to immunize twenty piglets without TGEV neutralizing antibodies, and use 200 μL of virus culture solution as a blank control. 4 times higher than that of spinner bottle culture, the results are shown in the following table.

Table 1 Comparison of the immune effects of virus cultured in bioreactors and in spinner bottles

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (5)

1. utilize bio-reactor to produce a method for transmissible gastro-enteritis virus, it is characterized in that, comprise the steps:

1) individual layer passage cell is prepared;

2) transmissible gastro-enteritis virus production kind of poison is prepared, specifically comprise the steps: with virus-culturing fluid transmissible gastro-enteritis virus kind poison to be inoculated in individual layer passage cell in 2-5% ratio to cultivate, gather in the crops virus liquid to during cell more than 80% pathology; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage rejuvenation, and the product that at every turn goes down to posterity carries out viral TCID ₅₀and Sterility testing, reach viral TCID ₅₀stablize and reach 10 ⁷tCID ₅₀rejuvenation is stopped, as production kind of a poison during/more than mL;

3) before cell suspension inoculation to bio-reactor, steril cell growth media is added by the 50-70% of bio-reactor cumulative volume, and add microcarrier by 4-10g/L concentration in often liter of steril cell growth media, start bio-reactor, 20-50 minute is stirred in the slow speed of revolution, by step 1) after the individual layer passage cell EDTA-trysinization liquid prepared digests, suspend with cell growth medium, being prepared into cell density is 1-3 × 10 ⁵the cell suspension of individual/mL, be inoculated in bio-reactor, absorption on the microcarrier of passage cell in bio-reactor is cultivated, described EDTA-trysinization liquid is be Hank ' the S liquid of the pancreatin of 0.05%-0.25% and the EDTA of 0.02% containing quality volume fraction, the formula of described cell growth medium is: massfraction is 95%-98%MEM liquid, 2%-5% bovine serum, final concentration is the antibiotic of 100-200IU/mL, pH value is 6.8-7.5, the parameter of bio-reactor setting is: culture temperature 35-38 DEG C, pH value 6.8-7.5, dissolved oxygen 50%-70%, stirring velocity 50-80rpm,

4) when passage cell grows to the 80%-90% of microcarrier, empty ball rate is lower than 5%, and full ball rate is greater than 80%, and cell counting reaches 3-5 × 10 ⁶individual/more than mL time, the inoculum size inoculation step 2 with 2-5%) prepare kind poison, carry out virus absorption cultivate;

5) when passage cell more than 80% pathology on microcarrier, results virus liquid.

2. method according to claim 1, is characterized in that, also comprises and to be carried out by microcarrier before microcarrier joined cell growth medium cleaning and the step of sterilizing, be followed successively by with PBS immersion microcarrier more than 3 hours; Microcarrier 3-5 time is cleaned with PBS; Microcarrier is soaked, 121 DEG C of steam sterilizing 20-50min with PBS.

3. method according to claim 1 and 2, is characterized in that, described passage cell is ST cell or vero cell.

4. method according to claim 1 and 2, is characterized in that, described transmissible gastro-enteritis virus is floating plumage vaccine strain, h-5 vaccine strain or magnificent strain.

5. method according to claim 1 and 2, it is characterized in that, described microcarrier is Gelatin based Macroporous Microcarriers, polystyrene microcarrier, PHEMA microcarrier, chitin microcarrier, polyurethane foam microcarrier, alginate jelly microcarrier, magnetic microcarrier, Cytodex1, Cytodex2, Cytodex3, Cytopore and/or Cytoline.