CN115896039A

CN115896039A - Method for producing rotavirus vaccine by using sheet-shaped carrier culture process

Info

Publication number: CN115896039A
Application number: CN202111051745.7A
Authority: CN
Inventors: 安祺; 田大勇; 阮俊程; 张雅春; 郭跃
Original assignee: Beijing Saierfusen Biotechnology Co ltd; Shanghai Qingsai Biotechnology Co ltd
Current assignee: Beijing Saierfusen Biotechnology Co ltd; Shanghai Qingsai Biotechnology Co ltd
Priority date: 2021-09-08
Filing date: 2021-09-08
Publication date: 2023-04-04

Abstract

The invention provides a method for producing rotavirus, in particular to a method for culturing the rotavirus in a bioreactor provided with a sheet-shaped carrier. The method avoids the influence of residual serum in the cell culture process on the pancreatic enzyme component in the rotavirus culture process through perfusion, determines the using concentration of the pancreatic enzyme and perfusion speed at different stages, finally performs the virus inoculation under the condition of high-density cells, and obtains the rotavirus harvest liquid with high titer and high yield. The problems of complicated manual operation, large workload, low virus titer and the like of a cell factory process in the prior rotavirus vaccine production are solved.

Description

Method for producing rotavirus vaccine by using sheet-shaped carrier culture process

Technical Field

The invention relates to the technical field of biological medicine and biology, in particular to a method for producing rotavirus vaccine by using a flaky carrier culture process.

Background

Rotavirus (RV) is a digestive tract virus with an extremely wide infection range; RV can be classified into 7 groups of A, B, C, D, E, F and G according to VP6 antigenicity (located in the capsid, highly conserved), and the groups A and B can infect humans, wherein the group A is the main pathogen causing infantile infection and severe diarrhea, the group B can infect adults and cause diarrhea, and the group C mainly causes sporadic diarrhea; groups D-G only infected animals.

RV vaccine immunization can remarkably reduce the proportion of severe diarrhea of infants caused by RV infection. The replication of most RV vaccine strains is pancreatic enzyme dependent, since the fusion protein VP4 of RV can be efficiently cleaved into VP8 with amino terminal and VP5 with carboxyl terminal under the action of pancreatic enzyme, and the two fragments play an important role in the early stage of virus invasion into host cells. The virus amplification process of RV becomes the core process in the production process of RV vaccine.

Most RV vaccine strains can cause cytopathic effect, and considerable RV virus particles exist in cells; in order to obtain more virus particles, the conventional RV cell factory production process and the conventional microcarrier bioreactor production process collect virus liquid and cells together after the virus culture is finished, and then break the cells by various technical means so as to release more RV virus particles into the obtained liquid. These processes result in impurities such as cellular proteins, nucleic acids and subcellular structures being present in the virus harvest in excess, which presents great technical difficulties for the later purification and needs to be removed as much as possible. On the one hand, the purification process is more complicated, and on the other hand, the quality control of the vaccine is further risked.

In addition, no matter the cell factory process or the microcarrier bioreactor process, the operation difficulty is high in the stage of removing the FBS in the cell culture stage, and the cells are easy to fall off in the cleaning process to influence the smooth operation of the whole virus culture process.

Therefore, there is a need in the art to develop a new rotavirus vaccine production method.

Disclosure of Invention

The invention aims to provide a convenient and efficient method for producing rotavirus vaccine.

In a first aspect of the present invention, there is provided a method for producing a rotavirus, comprising the steps of:

(a) Providing a bioreactor, wherein a sheet-shaped carrier is arranged in the bioreactor, the bioreactor is provided with a sample inlet and a sample outlet which can be opened and closed, and the working volume of the bioreactor is V0;

(b) The host cells were cultured at 5 x 10 ⁵ -1.5*10 ⁶ Inoculating the cells with the cell density of each cell/ml into the bioreactor, adding a first culture solution for culturing for 7-9 days, and starting perfusion culture from the 3 rd day of culture, thereby obtaining host cells attached to the sheet-shaped carrier with high cell density;

(c) Draining the culture supernatant of the host cells attached to the sheet-shaped carrier from a sample outlet, washing the host cells attached to the sheet-shaped carrier with a first washing solution, continuously perfusing the bioreactor with a volume of 6 × V0 of a second washing solution, draining the supernatant in the bioreactor, and adding a second culture solution into the bioreactor;

(d) Providing an activated rotavirus seed, inoculating the activated rotavirus into a second culture solution in the bioreactor, and inoculating with an MOI of 0.001-0.01, preferably 0.003;

(e) And after the virus inoculation is carried out for 12-16h, continuously perfusing the bioreactor by using a fresh second culture solution, and simultaneously collecting effluent liquid in a perfusion process to obtain virus harvest liquid containing the rotavirus, wherein the perfusion speed of the perfusion is v1, and the perfusion time is 6-8 days.

In another preferred example, the culture conditions of the bioreactor are set to 37 ℃, pH 7.2, DO 50% and stirring speed 100rpm.

In another preferred embodiment, the rotavirus is selected from the group consisting of: CDC-9, CDC-66, or a combination thereof.

In another preferred embodiment, the rotavirus is CDC-9.

In another preferred embodiment, the host cell is a Vero cell.

In another preferred embodiment, in step (b), the host cell is cultured at 5 x 10 ⁵ Cell density of one/ml was seeded into the bioreactor.

In another preferred example, in the step (b), the perfusion culture method comprises: adding fresh first culture solution from an injection port of the bioreactor, and simultaneously opening an outlet port to discharge culture supernatant of the host cells attached to the sheet-shaped carrier from the outlet port.

In another preferred example, in the step (b), the glucose concentration in the culture supernatant of the host cells attached to the sheet-like support during perfusion culture is not lower than 2g/L.

In another preferred example, in step (b), the glucose concentration in the reactor and the perfusate is measured every 24h during the perfusion culture process, so as to calculate the glucose consumption R, and the specific calculation formula is as follows:

wherein R is the unit glucose consumption per 24h of cells in the reactor, in g/L;

g0 is the glucose concentration of the culture solution in the reactor before 24h, and the unit is G/L;

g1 is the glucose concentration of the culture solution in the reactor, and the unit is G/L;

v0 is the working volume of the reactor in L;

g2 is the glucose concentration of the basic culture medium in G/L;

g3 is the glucose concentration in perfusion effluent, and the unit is G/L;

v2 is the volume of 24h perfusion effluent in L.

In another preferred example, in step (c), the washing method is: adding the first washing liquid from the sample inlet of the bioreactor, stirring for 5-15min, and discharging the washing liquid from the sample outlet.

In another preferred embodiment, in step (c), the number of washing is 2 to 4.

In another preferred example, in step (c), the perfusion method comprises: and adding a second washing solution from the sample inlet of the bioreactor, and simultaneously opening the sample outlet to discharge the supernatant in the bioreactor from the sample outlet.

In another preferred embodiment, in step (c), when perfusion is over, the BSA content of the supernatant in the bioreactor is not more than 100ng/ml, preferably not more than 50ng/ml.

In another preferred embodiment, in step (d), the activated rotavirus seed virus is prepared according to the following method:

adding rotavirus seed virus into activating solution containing pancreatin 15 μ g/ml and calcium chloride 800 μ g/ml, and activating at 37 deg.C for 1.0-2.0 hr to obtain activated rotavirus seed virus.

In another preferred example, in step (e), the perfusion method comprises: adding a fresh second culture solution from the sample inlet of the bioreactor, simultaneously opening the sample outlet, discharging the supernatant in the bioreactor from the sample outlet, controlling the speed of the inlet and outlet solution by a pump, and keeping the total volume of the culture solution in the bioreactor constant.

In another preferred embodiment, the first culture solution is a basal medium containing 8% -10% of FBS.

In another preferred example, the second culture solution is a basal culture medium containing 20-30. Mu.g/ml of pancreatin.

In another preferred example, the second culture solution is a basal culture medium containing 30. Mu.g/ml of pancreatin.

In another preferred embodiment, the first washing liquid is selected from the group consisting of: PBS buffer solution and a serum-free basic culture medium.

In another preferred embodiment, the second wash solution is a serum-free basal medium.

In another preferred embodiment, the first washing liquid and the second washing liquid are the same or different washing liquids.

In another preferred embodiment, the basal medium is selected from the group consisting of: DMEM medium, IMDM medium, M199, or a combination thereof.

In another preferred example, the basic culture medium contains glucose, and the concentration of the glucose is 3.9-4.5g/L.

In another preferred embodiment, in step (e), the method further comprises detecting the glucose concentration of the supernatant in the bioreactor every 12h after the rotavirus seed inoculation, and adjusting the perfusion speed v1 according to the detected glucose concentration to maintain the glucose concentration of the supernatant in the bioreactor to be not lower than 2g/L.

In another preferred example, in step (e), the perfusion rate V1= (0-4) × V0/day for the perfusion.

In another preferred embodiment, the titer of rotavirus in the virus harvest of rotavirus is not less than 6.25lgCCID ₅₀ /ml。

In another preferred embodiment, the method further comprises purifying the virus harvest containing the rotavirus after step (e), thereby obtaining a purified rotavirus.

In a second aspect of the invention, there is provided a rotavirus prepared by a process according to the first aspect of the invention.

In another preferred embodiment, the rotavirus is CDC-9.

In a third aspect of the present invention, there is provided a vaccine composition comprising: (a) a pharmaceutically acceptable carrier; and (b) a rotavirus prepared by a process according to the first aspect of the invention.

In another preferred embodiment, the vaccine composition further comprises: (c) an adjuvant.

In another preferred embodiment, the adjuvant is selected from the group consisting of: particulate and non-particulate adjuvants.

In another preferred embodiment, the particulate adjuvant is selected from the group consisting of: an aluminum salt, a water-in-oil emulsion, an oil-in-water emulsion, a nanoparticle, a microparticle, a liposome, an immunostimulatory complex, or a combination thereof;

in another preferred embodiment, the non-particulate adjuvant is selected from the group consisting of: muramyl dipeptide and its derivatives, saponin, lipid A, cytokine, derivative polysaccharide, bacterial toxin, microorganism and its products such as mycobacteria (mycobacterium tuberculosis, bacillus calmette-guerin), bacillus pumilus, bordetella pertussis, propolis, or their combination.

In a fourth aspect of the invention, there is provided the use of a rotavirus of the second aspect of the invention in the preparation of a rotavirus vaccine.

In another preferred embodiment, the rotavirus vaccine is an inactivated rotavirus vaccine or an oral rotavirus vaccine.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Figure 1 shows perfusion rates during the Vero cell culture phase.

FIG. 2 shows a glucose consumption curve during the culture phase of Vero cells.

FIG. 3 shows glucose metabolism during the virus culture phase.

FIG. 4 shows perfusion rates during the virus culture phase.

FIG. 5 shows a multi-step growth curve of RV virus under plate-like carrier bioreactor conditions.

Detailed Description

The present inventors have made extensive and intensive studies and, for the first time, have systematically developed a method for producing rotavirus in a bioreactor using a sheet-like carrier, thereby being useful for producing rotavirus vaccine. The invention solves the problem of the influence of residual serum in the cell culture process on the pancreatin component in the virus culture process through perfusion, determines the glucose metabolism condition and the growth cycle of the Vero cells in the sheet carrier culture method, determines the rotavirus virus inoculation time, the pancreatin use concentration and the perfusion speed in different stages after virus inoculation, and finally obtains the rotavirus vaccine with high titer and high yield by virus inoculation under the condition of high-density cells. Solves the problems of complicated manual operation, large workload, low virus titer and the like in the cell factory process in the prior rotavirus vaccine production.

On the basis of this, the present invention has been completed.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. And other definitions are set forth throughout this application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Bioreactor

The invention provides a method for producing rotavirus by using a flaky carrier in a bioreactor, the bioreactor is provided with a fixed bed basket type stirring system, the flaky carrier can be fixed in a packed bed, the surface area is provided for the adherent growth of animal cells, parameters such as pH, DO, temperature, stirring speed and the like can be controlled, and a stable and proper growth environment is provided for the cells and the virus.

In the present invention, the bioreactor is provided with a sheet-like support fixed in a packed bed, and the culture conditions of the bioreactor are set to 37 ℃ temperature, pH 7.2, DO 50%, stirring speed 100rpm.

Sheet-like support

The invention provides a method for producing rotavirus by using a sheet-shaped carrier in a bioreactorAccording to the method, the main component of the flaky carrier is polyester fiber, and the structure is a multi-gap net structure, so that the adherent culture of animal cells is facilitated. Can provide about 1200cm per gram of sheet-shaped carrier ² Surface area of (2) satisfies 3 x 10 ⁸ The growth space required by the Vero cells is 2-3 times that of common microcarriers such as Cytodex 1, and the flaky carriers can be round sheets, three-dimensional page types and the like. For example, in a preferred embodiment of the present invention, the sheet-like carrier is a fibrous-Cel Disks.

Rotavirus virus

The present invention provides a rotavirus prepared by a process according to the first aspect of the invention. The rotavirus may be from any source so long as it is capable of propagating in cells susceptible to rotavirus. In the present invention, the rotavirus may be a wild type, or a subtype thereof, or a mutant thereof or an attenuated strain thereof, or the like. For example, in a preferred embodiment of the invention, the rotavirus is RV strain CDC-9 of G1P8 type.

The rotavirus production method of the invention

The rotavirus production method of the invention has a plurality of advantages compared with the existing rotavirus production method. Specifically, the method of the invention enables the inoculated host cells to be attached to the flaky carrier to grow through configuring the flaky carrier in the bioreactor, and compared with the host cells directly attached to the flaky carrier to grow in an adherent manner in the bioreactor, the host cells attached to the flaky carrier are more difficult to fall off; moreover, after the rotavirus is inoculated, continuous perfusion culture is realized through the bioreactor, and FBS residue caused by early-stage host cell culture in a culture system is reduced, so that the culture system is more beneficial to the growth of the rotavirus depending on high-concentration pancreatin.

Specifically, in a preferred embodiment of the present invention, the rotavirus production method comprises the following steps:

(a) Providing a bioreactor, wherein a sheet-shaped carrier is arranged in the bioreactor, the bioreactor is provided with a sample inlet and a sample outlet which can be opened and closed, and the volume of the bioreactor is V0;

(b) Vero cells were plated at 5 x 10 ⁵ -1.5*10 ⁶ (ii) inoculating the individual cells/ml at a cell density into the bioreactor, and adding a DMEM medium containing 8% fbs for culture for 8 days, thereby obtaining high-density Vero cells adhered to the sheet-shaped carrier;

(c) Draining the culture supernatant of the Vero cells attached to the sheet-shaped carrier from a sample outlet, washing the host cells attached to the sheet-shaped carrier for 3 times by using PBS buffer solution, continuously perfusing the bioreactor by using 6 x V0 volume of serum-free DMEM medium, and adding DMEM medium containing 30 mu g/ml pancreatin into the bioreactor after emptying the supernatant in the bioreactor;

(d) Inoculating activated rotavirus CDC-9 virus into DMEM medium containing 30 μ g/ml pancreatin in the bioreactor at MOI = 0.003;

(e) And (3) continuously perfusing the bioreactor by using fresh DMEM culture solution containing 30 mu g/ml pancreatin 16 hours after virus inoculation, and collecting effluent liquid in a perfusion process to obtain virus harvest liquid containing the rotavirus CDC-9, wherein the perfusion speed of perfusion is v1, and the perfusion time is 6 days.

Wherein, in step (e), the perfusion rate V1= (0-4) × V0/day of the perfusion. The perfusion speed v1 is used for detecting the glucose concentration of the supernatant in the bioreactor every 12h after rotavirus seed inoculation, and adjusting the perfusion speed v1 according to the detected glucose concentration to maintain the glucose concentration of the supernatant in the bioreactor to be not lower than 2g/L.

Prepared rotavirus and vaccine composition

The invention also provides a high purity rotavirus prepared by the method of the invention and a vaccine composition containing the rotavirus.

The rotavirus prepared by the invention has less impurities and high titer by determination, and the virus titer in the rotavirus harvest liquid before the purification step can reach 7.5lg CCID ₅₀ /ml。

The rotavirus (inactivated) prepared by the invention can be used as immunogen for stimulating animals to generate immune response aiming at the rotavirus, thereby protecting the animals (human) from being infected by the rotavirus.

One preferred composition is a prophylactic vaccine composition. The vaccine composition of the present invention may be a monovalent or multivalent vaccine composition.

These vaccines comprise the high purity, high titer rotavirus prepared in accordance with the invention, and are typically combined with "pharmaceutically acceptable carriers" which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive viral particles. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants").

Preferred adjuvants that enhance the effect of the composition include, but are not limited to: squalene, muramyl dipeptide, MF59, AS03, monophosphoryl lipid A, flagellin, cpG-ODN, poly (I: C), and small molecules of aluminum or calcium salts.

The vaccine compositions of the invention (including rotaviruses, pharmaceutically acceptable carriers and/or adjuvants) will generally contain diluents such as water, saline, glycerol, ethanol and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.

More particularly, vaccines, including immunogenic compositions, comprise an immunologically effective amount of rotavirus, as well as the other desirable components described above. An "immunologically effective amount" refers to an amount that is effective for treatment or prevention in a single dose or in a continuous dose administered to an individual. The amount will depend on the physiological condition of the animal (e.g., human), the ability of the immune system to synthesize antibodies, the degree of protection desired, and other relevant factors.

In the present invention, the vaccine composition or immunogenic composition can be prepared into an injectable agent, such as a liquid solution or emulsion; it can also be made into solid form suitable for being mixed with solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes, enhancing the adjuvant effect in the pharmaceutically acceptable carrier described above.

The conventional approach is to administer immunogenic compositions by injection from the parenteral (subcutaneous or intramuscular) route. Other formulations suitable for other modes of administration include oral and transdermal applications, and the like. The therapeutic dose may be a single dose regimen or a multiple dose regimen. The vaccine compositions of the present invention may be administered in combination with other immunomodulators.

The main advantages of the invention are:

(1) The method of the invention uses the sheet-shaped carrier to culture the cells, on one hand, the total number of the cells in the culture space of unit volume in the bioreactor can be more, and the method is also beneficial to the improvement of the virus titer.

(2) On the other hand, due to the material and structural characteristics of the sheet-shaped carrier, the cells can still be tightly attached without falling after the FBS is cleaned, the RV is inoculated and the high-concentration pancreatin is used for culture, so that the cell source impurities caused by cell breakage in the virus harvesting liquid can be greatly reduced at the later stage.

(3) The actual density of the cells in the culture space is larger, and the cells do not fall off in the virus culture process, so the number of the viruses released into the supernatant is larger, the harvest liquid with higher virus titer can be obtained without crushing the virus-inoculated cells, and the process is simplified.

(4) Because the cell shedding proportion is small, the method can continuously perfuse a fresh cell culture medium in the virus culture process, the cell state can be better maintained, and finally more virus liquid with higher virus titer can be obtained.

(5) Different from a cell factory process and a microcarrier bioreactor process, the process has low cell shedding rate in the virus culture process, and the impurities of cell fragments in a virus harvesting solution are less; no matter the inactivated rotavirus vaccine or the oral rotavirus vaccine is developed, the complexity of the purification process is obviously reduced, the quality of the vaccine stock solution is higher, and the production and the development of the high-quality vaccine are more facilitated.

Example 1

Vero cell inoculation bioreactor and perfusion culture method development

After digesting sufficient Vero cells into single cells, add 5X 10 ⁵ Cells/ml cell density was inoculated into 8% fbs-containing DMEM medium in a sheet-like carrier bioreactor; then, the culture conditions of the bioreactor were set at 37 ℃ and pH 7.2, DO 50%, and the stirring speed was 100rpm.

And comprehensively monitoring multiple indexes in the culture process. Samples were taken from the jar every day 2 days before culture and glucose concentration was measured using a Roche glucometer. Perfusion of fresh medium (DMEM medium with 8% FBS) starting on day 3 of culture, increasing the perfusion rate day by day according to the nutrient demand of the cells in the tank until day 7 (VVD means the perfusion volume, for example the bioreactor used is a 3.75L working volume, in perfusion, if the perfusion volume is 3.75L, it is noted as 1 VVD), with the aim of determining the cell growth curve under these conditions and determining the exact time point for later virus inoculation. During perfusion culture, the volume of the tank body discharge liquid and the glucose concentration are counted besides sampling in the tank body, the glucose concentration in a growth period is ensured to be not lower than 2g/L, and the daily glucose metabolism is analyzed.

As a result, as shown in FIG. 1, the amount of the perfused medium was gradually increased as the culture proceeded, and was not significantly increased any more on day 7. Meanwhile, the results of the measurement of glucose in the tank of the bioreactor also showed that the sugar consumption did not increase significantly by day 8, and was almost the same as that of day 7 (fig. 2). This indicates that Vero cells based on this condition reached plateau starting on day 7.

Example 2

FBS cleaning condition groping

CDC-9 is a virus that requires high concentrations of pancreatin (20-30. Mu.g/ml) for efficient culture, which requires strict control of factors affecting the action of pancreatin, FBS being the most critical one of these factors. 7-9 days after the Vero cell is inoculated and cultured (or when the total sugar consumption in a reactor is more than 2.5 g/L), in order to remove the FBS in the Vero cell culture process to the maximum extent, firstly discharging the culture medium in the tank as much as possible, injecting sterilized PBS, stirring for 10min, then emptying the liquid in the tank, injecting PBS again, repeating the steps for 3 times, and removing most of the FBS; then, perfusion with serum-free DMEM medium was started, and the perfusion rate was 8VVD/d. Sampling and detecting the BSA concentration in the reactor by 2 tanks per perfusion.

Results as shown in table 1, when the perfusion volume of DMEM medium reached 4VVD, the BSA content in the reactor tank was already below 100ng/ml; when the perfusion volume reached 6VVD, the BSA content was even lower than 50ng/ml.

TABLE 1 BSA content in Wash and perfusion treatment reactor

Treatment method	BSA content (ng/ml)
		Cell culture medium	1613000
PBS wash 3 times	3035
		Perfusion 2VVD	147
Perfusion 4VVD	97
		Perfusion 6VVD	25

Example 3

Investigation of perfusion conditions under high concentration pancreatin conditions

In order to verify the maintenance of cells in a sheet-like carrier bioreactor under serum-free and high-concentration pancreatin conditions (30. Mu.g/ml), porcine pancreatin was added to the culture medium to a final concentration of 30. Mu.g/ml after the BSA concentration in the culture medium in the reactor tank was kept below 100ng/ml; the cells were then monitored for sugar consumption curve and cell number in the perfusion effluent.

As a result, it was found that after perfusing the culture medium containing pancreatin, the sugar consumption in the tank began to decrease significantly with time, but there was no significant cell shedding in the effluent from the tank until day 7 of the perfusion, the sugar consumption had decreased by 80% from day 1, no significant cell shedding was detected in the effluent, and the clarity of the liquid was still high (see Table 2). This demonstrates that this protocol possesses the potential for use in the CDC-9 sheet support bioreactor culture process.

TABLE 2 cell metabolism and exfoliation following replacement of pancreatin-containing maintenance fluid

Perfusion time/d	Cell sugar consumption g/L	Cell shedding amount/d
			0	3.04	0
1	1.31	2.1*10 ⁶
			2	0.92	1.5*10 ⁷
3	0.84	8.3*10 ⁶
			4	0.72	3.7*10 ⁶
5	0.67	2.2*10 ⁶
			6	0.61	1.3*10 ⁶
7	0.59	1.1*10 ⁶

The above results demonstrate that FBS can be conveniently removed from the tank body by perfusion. And the replacement of the culture medium inside the bioreactor of the sheet-like carrier does not have a significant effect on the cells, the proportion of cell shedding is less than 0.1% (as can be seen by counting the cells in the perfusion effluent), and the cell status is affected only to a very limited extent during the perfusion wash.

Example 4

Inoculation and perfusion culture of RV

After digesting sufficient Vero cells into single cells, add 5X 10 ⁵ Inoculating the cells per ml into a sheet-shaped carrier bioreactor; then, the culture conditions of the bioreactor were set at 37 ℃ and pH 7.2, DO 50%, and the stirring speed was 100rpm. Discharging culture medium from the tank when the glucose metabolism in the bioreactor is more than 2.5g/L, injecting sterilized PBS, stirring for 10minEmptying the liquid in the tank, filling PBS again, repeating the process for 3 times, and removing most of FBS; perfusion with serum free DMEM was then continued for 6 tank volumes to wash out the residual FBS sufficiently. Finally, the liquid in the tank was emptied and replaced with a serum-free DMEM maintenance solution containing high concentrations of pancreatin (30. Mu.g/ml).

CDC-9 requires a high pancreatin concentration, and therefore, the virus inoculation MOI =0.003, and pancreatin and calcium chloride were supplemented to a final pancreatin concentration of 15. Mu.g/ml and a final calcium chloride concentration of 800. Mu.g/ml, and the mixture was put in a 37 ℃ water bath and activated for 1.5 hours, and then inoculated into a DMEM holding solution containing high-concentration pancreatin (30. Mu.g/ml) in a reactor.

Monitoring of sugar consumption shows that cell metabolism is accelerated and glucose concentration in maintenance liquid is obviously reduced after 12-16h of virus inoculation under the condition of MOI =0.003, and DMEM maintenance liquid containing 30 mu g/ml pancreatin is perfused at the moment. After virus inoculation, the perfusion speed is adjusted according to the glucose concentration reduction speed in the tank body, and the growth curve of the virus is drawn by detecting the virus titer in the effluent liquid.

The sugar consumption and perfusion profiles throughout the virus culture phase are shown in fig. 3 and 4: after virus inoculation, the sugar consumption of the cells begins to rise rapidly and reaches 6.13g/L at most within 24 hours, then the consumption of glucose by the cells begins to decrease rapidly, the perfusion speed is reduced, after virus inoculation for 168 hours, the sugar consumption is almost zero, and the perfusion is stopped.

The research result on the RV growth characteristics shows that the virus can still rapidly amplify the progeny virus even under the condition of low MOI (figure 5), and the titer high value reaches 7.5lg CCID ₅₀ Per ml; even at 8 days after inoculation, the virus titer in the supernatant could still reach 6.25lgCCID without any value for sugar consumption ₅₀ /ml。

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

1. A method for producing a rotavirus, the method comprising the steps of:

(b) The host cells are cultured at 5 x 10 ⁵ -1.5*10 ⁶ Inoculating the cells with the cell density of each cell/ml into the bioreactor, adding a first culture solution for culturing for 7-9 days, and starting perfusion culture from the 3 rd day of culture, thereby obtaining host cells attached to the sheet-shaped carrier with high cell density;

2. The method of claim 1, wherein the rotavirus is selected from the group consisting of: CDC-9, CDC-66, or a combination thereof.

3. The method according to claim 1, wherein in step (b), the glucose concentration in the culture supernatant of the host cells attached to the sheet-like support during perfusion culture is not less than 2g/L.

4. The method according to claim 1, wherein in step (c), the supernatant in the bioreactor has a BSA content of no greater than 100ng/ml, preferably no greater than 50ng/ml, when perfusion is complete.

5. The method of claim 1, wherein the first culture fluid is a basal medium comprising 8% -10% fbs.

6. The method of claim 1, wherein the second culture fluid is a basal medium containing 20-30 μ g/ml pancreatin.

7. The method according to claim 1, wherein in step (e), the perfusion rate V1= (0-4) × V0 per day for perfusion.

8. A rotavirus prepared by the process of claim 1.

9. A vaccine composition, comprising: (ii) (a) a pharmaceutically acceptable carrier; and (b) a rotavirus prepared by the method of claim 1.

10. Use of the rotavirus of claim 2 in the preparation of a rotavirus vaccine.