CN116836944A - Rotavirus vaccine and method for producing rotavirus vaccine - Google Patents

Rotavirus vaccine and method for producing rotavirus vaccine Download PDF

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CN116836944A
CN116836944A CN202310698910.0A CN202310698910A CN116836944A CN 116836944 A CN116836944 A CN 116836944A CN 202310698910 A CN202310698910 A CN 202310698910A CN 116836944 A CN116836944 A CN 116836944A
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rotavirus
cells
pancreatin
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宋俐霏
孟凡红
姚志东
栾春芳
樊艳明
沙雪艳
陈飞
陈娜
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Sinovac Research & Development Co ltd
Sinovac Dalian Vaccine Technology Co ltd
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Sinovac Dalian Vaccine Technology Co ltd
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Abstract

The present disclosure relates to a rotavirus vaccine and a method of producing a rotavirus vaccine. The method for producing rotavirus vaccine comprises inoculating rotavirus into cell culture solution containing cells loaded on microcarrier; in the process of maintaining the concentration of pancreatin at 2-10 mg/L and Ca 2+ At a concentration of 1mM or more, to collect a rotavirus-containing supernatant. The method for producing rotavirus vaccine can obtain rotavirus with high titer, so that the harvest liquid does not need to be concentrated, and the rotavirus caused by concentration is avoidedTiter loss.

Description

Rotavirus vaccine and method for producing rotavirus vaccine
Technical Field
The present disclosure belongs to the field of biological medicine preparing technology, and is especially one kind of rotavirus vaccine and its production process.
Background
Rotavirus infection is the main cause of severe dehydration diarrhea in infants under 5 years old. Rotavirus vaccines are the primary method of preventing rotavirus infection. Human immunity to rotavirus is mainly through stimulation of intestinal immunity to secrete IgA. The subunit and inactivated vaccine can only be immunized by injection, but the injection cannot excite the intestinal secretion IgA, so that the protection against diarrhea caused by rotavirus cannot be provided. The oral attenuated live rotavirus vaccine is the only rotavirus vaccine on the market at present.
The high-concentration and high-quality rotavirus is the basis for producing attenuated live vaccines, inactivated vaccines, genetic engineering vector vaccines and multivalent or combined vaccines which take rotavirus attenuation and derivatives thereof as main components, so that the preparation process of the high-efficiency rotaattenuated live vaccines suitable for industrialization has great economic and social values. However, none of the existing attenuated rotavirus vaccine processes meet the above requirements.
Therefore, there is a need to provide a culture method capable of obtaining rotaviruses with high titer.
Disclosure of Invention
In order to solve the technical problems, the present disclosure provides a method for producing rotavirus vaccine.
In a first aspect, the present disclosure provides a method of producing a rotavirus vaccine, the method comprising:
inoculating rotavirus into a cell culture solution, wherein the cell culture solution contains cells loaded on microcarriers;
in the process of maintaining the concentration of pancreatin at 2-10 mg/L and Ca 2+ At a concentration of 1mM or more, to collect a rotavirus-containing supernatant.
In some embodiments, the concentration of pancreatin is maintained in the range of 2 to 8 mg/L.
In some embodiments, the Ca 2+ The concentration of (2) is kept in the range of 1 to 2 mM.
In some embodiments, by supplementing pancreatin and/or Ca 2+ Maintains pancreatin and/or Ca 2+ Is a concentration of (3).
Adding pancreatin and Ca 2+ Is to maintain pancreatin and Ca in the culture solution 2+ Is not lower than its initial concentration to increase rotavirus titer.
Supplement of Ca 2+ When using Ca in calcium salt 2+ Is a meter of the substance of (a).
In some embodiments, ca is maintained by the addition of calcium salts 2+ Concentration. The calcium salt is selected from calcium phosphate, calcium chloride, calcium sulfate, calcium nitrate, calcium lactate, calcium gluconate, calcium citrate, calcium succinate, and amino acid chelated calcium or a combination thereof.
In some embodiments, the pancreatin is fed continuously at a rate of no more than 400 μg/L/h, or fed in batches.
In some embodiments, ca is added 2+ In such a way that Ca is continuously added at a rate of not more than 0.6 mM/day 2+ 。
In some embodiments, the rotavirus has a multiplicity of infection MOI of 0.001 to 0.1.
In some embodiments, the rotavirus includes, but is not limited to, human rotavirus, simian rotavirus, bovine rotavirus, rabbit rotavirus, porcine rotavirus, equine rotavirus, canine rotavirus, goat rotavirus, avian rotavirus, murine rotavirus, and the like.
In some embodiments, rotaviruses include, but are not limited to, human-bovine reassortant rotaviruses, human-ovine reassortant rotaviruses, or human-porcine reassortant rotaviruses.
In some embodiments, the rotavirus is selected from the group consisting of a G9 rotavirus, a G1 rotavirus, a G2 rotavirus, a G3 rotavirus, a G4 rotavirus, and a P1A 8 rotavirus.
In some embodiments, rotavirus may be inoculated directly into the culture broth without activation.
In some embodiments, the culture broth is not discharged during amplification of rotavirus.
In some embodiments, the volume of the culture broth is 2-1000L. For example, rotavirus is amplified in at least 2L, at least 5L, at least 20L, at least 50L, at least 100L, at least 200L, at least 300L, at least 500L, at least 800L or at least 1000L of culture broth.
In some embodiments, the serum concentration of the culture broth is less than or equal to 0.05%, preferably the culture broth is serum free.
In some embodiments, the culture fluid is selected from, but not limited to 199 medium, MEM medium, DMEM medium, and M199 medium.
In some embodiments, the culture broth further comprises one or more of glucose, glutamine, non-essential amino acids, and growth factors.
In some embodiments, during amplification of rotavirus, at least one of cells, glucose, glutamine, nonessential amino acids, growth factors, or basal medium loaded on the microcarrier is also supplemented.
In some embodiments, the growth factor comprises an epidermal growth factor and/or a fibroblast growth factor.
In some embodiments, the method further comprises: the cells are cultured in a medium containing the microcarriers, preferably such that the cells are grown full of microcarriers.
In some embodiments, the cell culture conditions are at 35-38deg.C, pH 7.0-7.4, and dissolved oxygen of 35% -50%.
In some embodiments, the conditions of the cell culture further comprise agitation, preferably 40 to 100rpm.
In some embodiments, the cells are cultured in a liquid change culture.
In some embodiments, the plating is performed after a period of time, e.g., 24 hours, of the cell culture using a medium having a serum content no higher than the serum content of the initial medium.
In some embodiments, the plating is performed by removing a working volume, e.g., 50% -75% of the working volume of the broth supernatant, and then adding an equal volume of fresh medium, i.e., a sedimentation plating.
In some embodiments, the liquid-changing culture is performed by adopting a continuous perfusion mode, discharging the original culture liquid supernatant, and simultaneously supplementing a new culture medium, namely, perfusion liquid-changing culture.
In some embodiments, the cells are seeded at a density of 0.2X10 s in the medium 6 ～1.0×10 6 Individual cells/mL.
In some embodiments, the microcarriers are used in an amount of 1 to 10g/L.
In some embodiments, the microcarrier is selected from a porous structure based on dextran, collagen, plastic, gelatin, and cellulose.
In some embodiments, the microcarrier is selected from the group consisting of CytodexCytpdex/> 3D/>And Cytopore->
In some embodiments, the microcarrier is selected from the group consisting of CytodexAnd 3D->
In some embodiments, the cell is selected from the group consisting of Vero cells, MA-104 cells, MRC5 cells, GBK cells, 293 cells, BHK cells, and CHO cells.
In some embodiments, the method further comprises homogenizing and filtering the supernatant to obtain rotavirus liquid.
In some embodiments, the homogenizing is at a temperature of-5 to 15 ℃ and a pressure of 50 to 500bar.
In some embodiments, the microfiltration uses a filter membrane with a pore size of 0.2 to 1 μm.
In some embodiments, the method does not include: the supernatant was concentrated.
In some embodiments, the method further comprises: mixing the rotavirus liquid with a protective agent.
In some embodiments, the protectant includes 20-60% saccharide, 0.05-0.5 mol/L phosphate, and 0.05-0.5 mol/L carboxylate.
In some embodiments, the saccharide is selected from sucrose, lactose, dextran, trehalose, galactose, sorbitol, and mannose or a combination thereof.
In some embodiments, the carboxylate is selected from citrate and/or succinate.
In some embodiments, the ratio of rotavirus liquid to the protectant is 1:1 to 1:10.
In a second aspect, the present disclosure provides a rotavirus vaccine prepared by the method provided in the first aspect.
The rotavirus with the titer of more than 7.6 can be obtained by the method for producing the rotavirus vaccine, which satisfies the immune effect of the titer of more than 6.8 after hexavalent reconstitution, so that the harvest liquid does not need to be concentrated, and the rotavirus titer loss caused by concentration is avoided.
The rotavirus prepared by the method provided by the disclosure has the advantages of high titer and high quality.
The method for culturing rotavirus provided by the disclosure can harvest high-titer rotavirus without cell disruption.
The method for producing rotavirus vaccine provided by the disclosure not only does not need to concentrate culture supernatant, but also adopts a homogenization and microfiltration process in a downstream process section after harvesting the culture supernatant, so that the loss of rotavirus titer can be reduced, and the process is simple and low in loss. The rotavirus electron microscope results show that the rotavirus has high integrity. Therefore, the method for producing rotavirus vaccine provided by the present disclosure has simple process and is easy to industrialize.
The rotavirus vaccine provided by the disclosure has acid resistance and stability, and maintains the immunogenicity of rotavirus antigens.
Drawings
FIG. 1 shows the change in titer of the supernatant of rotavirus culture obtained in example 1.
Figure 2 shows the rotavirus titer and turbidity changes in the feed solution obtained in each process stage of example 1.
FIG. 3 shows an electron micrograph of rotavirus obtained in example 1.
Figure 4 shows the variation of rotavirus titer in the feed solution obtained in each process stage of example 2.
FIG. 5 shows the variation of rotavirus titer before homogenization and after homogenization at different temperatures in example 5.
Figure 6 shows the antacid and stabilizing effects of the different formulations of protectants of example 6 on rotavirus. In fig. 6, a shows the antacid effect of the different formulations of the rotaimmunogenic composition and B shows the stabilizing effect of the different formulations of the rotaimmunogenic composition.
Detailed Description
The present disclosure is further illustrated by the following examples, it being understood that the examples of the present disclosure are provided by way of illustration only and not by way of limitation, and that simple modifications of the present disclosure, while remaining within the spirit of the present disclosure, are intended to be within the scope of the appended claims.
Definition of the definition
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the meaning commonly understood by one of ordinary skill in the art. It should be noted that the terms used herein should be construed to have meanings consistent with the context of the present specification and should not be construed in an idealized or overly formal manner.
The expression "about" as used herein is as understood by a person of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art does not know the use of this term depending on the context in which it is used, "about" will mean that a particular value is up to plus or minus 10%.
The term "Rotavirus" (RV) as used herein is a double-stranded RNA (dsRNA) virus belonging to the Reoviridae (Reoviridae) Rotavirus (Rotavirus). Rotavirus is one of the main pathogens causing acute gastrointestinal infectious diseases of human newborns and various young animals such as pigs, cattle, sheep and the like, and clinical manifestations are mainly characterized by vomiting, diarrhea and dehydration.
Rotavirus is a non-capsule, icosahedral, with a diameter of about 70nm, and is a particle with short fiber and smooth outer edge similar to a wheel shape when observed by electron microscope. The virus particle is composed of a 3-layer structure (an outer layer capsid, an inner layer capsid and a core), 11 double-stranded ribonucleic acid fragments are wrapped in the middle, 6 structural proteins (VP 1-VP4, VP6 and VP 7) and 5 non-structural proteins (NSP 1-NSP 5) are encoded, and VP1-VP3 is marrow core protein. NSP4 has enterotoxin effect, is an important factor for diarrhea, VP4 (P protein) and VP7 (G protein) are specific antigen types, can induce neutralizing antibody to generate, and plays an immunoprotection role. VP6 has a group-specific epitope. RV is divided into groups (groups) of A-G7 according to antigenicity, A, B, G groups (groups) can cause zoonosis, other groups (groups) mainly cause diarrhea of animals, and a small number of infected people. Group A is the most common pathogen of diarrhea in children less than or equal to 5 years old, and can cause 90% of RV diarrhea. The VP7 antigen can be classified into 14 different G serotypes and 18P serotypes, with the G1, G2, G3, G4 and G9 serotypes being the most common among the population. During the proliferation of the virus, there may be recombination of 11 gene fragments, even between animal and human strains, which is a partial cause of a large number of different strains in nature. Rotaviruses infecting humans mostly belong to group A, and it is further possible to divide rotaviruses into 2 subgroups of 23G (G1-G23) serotypes (depending on VP7 neutralizing antigens) and 31P (P1-P13) serotypes (depending on VP4 hemagglutinin antigens). The G-P combination serotype is determined by the VP7 and VP4 of the virus, and currently, the rotaviruses of the G1P 8, G2P 4, G3P 8, G4P 8 and G9P 8]5 serotypes are the most important strains for human health.
The term "vaccine" as used herein is intended to include prophylactic or therapeutic vaccines. A prophylactic vaccine is one that is administered to stimulate an immune response to an antigen, so if an individual is subsequently exposed to an antigen, the preformed immunity will protect the individual from the corresponding disease associated with the antigen. A therapeutic vaccine is administered to an individual already suffering from an antigen-related disease, wherein the vaccine may elicit an immune response to the antigen or boost the existing immunity of the individual to the antigen to treat and/or ameliorate the symptoms of the disease.
The term "inactivated vaccine" as used herein is a virus or bacteria that is first cultured and then physically or chemically killed to render the whole virus or bacteria infectious and avirulent. Inactivated vaccines may consist of either whole viruses or bacteria or of split fragments thereof as split vaccines.
The term "rotavirus vector vaccine" as used herein refers to an immune composition having any one or more of rotavirus inner or outer shell skeleton genes VP4, VP6 and VP7 genes and gene edits thereof, which is obtained using genetic engineering means or a reconstitution method, with the virus or pseudovirus as an immunogen.
The term "amino acid chelated calcium" as used herein refers to an organic calcium species produced by chemical synthesis of a plurality of amino acids with an inorganic calcium salt.
The term "multiplicity of infection" (multiplicity of infection, MOI) as used herein is used to determine the phage titer of a host bacterium, referred to as indicator bacterium, and the ratio of the number of phage to indicator bacterium cells at the time of infection is referred to as the multiplicity of infection, i.e., the number of phage infected per cell.
The term "sedimentation exchange" as used herein means stopping the stirring of the reactor, discharging a certain amount of supernatant after the cells and carriers in which the cells grow settle to the bottom of the reactor, replenishing the culture solution or other solution, and restarting the stirring.
The term "perfusion changing liquid" as used herein means discharging a solution in a tank while feeding a culture liquid or other solution into a reactor without stopping stirring of the reactor. In the present disclosure, cells and microcarriers on which they are present are retained by special means within the reactor (e.g., by adding a rotating screen at the drain) with little or no cell exchange fluid from the reactor.
As used herein, controlling the serum concentration in the broth refers to calculating the remaining serum in the reactor without accounting for serum loss by counting the amount of serum in the influent and the amount of serum in the effluent, and thus estimating the theoretical serum concentration.
The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Examples toxic species: human bovine reassortant rotavirus seed was from NIH (National Institutes of Health national institutes of health)
MVS-BRV-1: human-bovine rotavirus G1 serotype
MVS-BRV-4: human-bovine rotavirus G4 serotype
MVS-BRV-10: human-bovine rotavirus G9 serotype
The rotavirus working virus seed is amplified and subcultured on the basis of NIH belly.
EXAMPLE 1 G9 Rotavirus culture and preparation of viral supernatant
1) According to 8X 10 5 cell/mL seeding Density Vero cells were seeded into 50L stirred bioreactor with 1g/L Cytodex1 microcarrier and 199 medium with 10% serum, cultured at 35℃at 100rpm, pH7.0 with dissolved oxygen at 50%. The microcarriers were grown for 4 days with a 199 medium sedimentation exchange of 75% working volume containing 2% serum every 24 hours.
2) The medium was allowed to settle and change 5 times with serum-free 199 medium.
Rotavirus type G9, pancreatin and CaCl 2 Respectively, into the reactor. In the reactor, according to the cell count result, G9 type rotavirus MOI=0.05, the final concentration of pancreatin is 2mg/L, caCl 2 The final concentration was 2mmol/L, and the culture was carried out at pH7.0, 60rpm and 36℃for 48 hours. During the period, 1mg/L pancreatin was added at 18 hours and 30 hours, respectively. During which samples were taken to detect the titer of the viral culture supernatant. The highest titer of rotavirus culture supernatant was found to be about 8.9lg CCID 50 the/mL is shown in FIG. 1.
3) Rotavirus culture supernatant was harvested by sedimentation. The rotavirus culture supernatant was homogenized at low temperature, the chiller temperature was set at 4 ℃, the pressure was 200bar, and the flow rate was 40L/h. The homogenizer effluent was filtered through a 1 μm filter and then through a 0.2 μm microfiltration membrane. As can be seen from fig. 2, the downstream process did not significantly change the viral titer, but the turbidity of the feed solution was significantly reduced.
4) The rotavirus obtained after filtration was observed using an electron microscope and the obtained rotavirus particles were found to be intact, to have a distinct spike structure and to be regularly ordered (fig. 3).
EXAMPLE 2 G1 rotavirus reactor culture and preparation of viral supernatant
1) According to 4X 10 5 cell/mL seeding Density MA-104 cells were seeded into 10L basket style bioreactor (NBS Co.) with 10g/L Cytodex1 microcarriers, 8% serum 199 Medium, 38℃40rpm, pH7.4Culturing with dissolved oxygen of 35%. The microcarriers were grown for 5 days from day 2 after inoculation of the cells by sedimentation with 199 medium containing 2% serum and 75% working volume each day.
2) The medium was subjected to sedimentation and 4-time exchange with serum-free 199 medium containing 1mmol/L of calcium salt, at which time the theoretical serum residue was 0.0078%.
3) Based on the cell count, rotavirus G1, 199 medium, pancreatin and CaCl were used 2 The solution was mixed and added to the reactor. In the virus culture solution in the reactor, according to the cell count result, G1 type rotavirus MOI=0.01, final concentration of pancreatin 2mg/L, caCl 2 The final concentration was 1mmol/L, and the culture was carried out at pH 7.2, 60rpm and 37℃for 60 hours. During this period, the sterile pancreatin solution was continuously fed using peristaltic pumps at a flow rate of about 200. Mu.g/L/h. The titer of the supernatant of the virus culture was about 8.2lg CCID 50 /mL。
4) Rotavirus culture supernatant was harvested by sedimentation. The rotavirus culture supernatant was passed through a 5 μm microfiltration membrane and homogenized at low temperature with a chiller temperature set at 2℃and a pressure of 500bar. The homogenizer effluent was filtered using a 0.45 μm microfiltration membrane. The results of virus titers after homogenization and filtration with 0.45 μm microfiltration membrane after 24h, 48h and 60h incubation in virus culture are shown in FIG. 4. From the results shown in fig. 4, it is clear that homogenization and filtration with a membrane in the downstream process stage have little effect on viral titer and substantially no loss of viral titer.
EXAMPLE 3 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 5L stirred bioreactor with 1 g/L3D TableTrix microcarriers, 5% serum 199 medium, 37℃at 80rpm, pH 7.2, dissolved oxygen 50% culture. From day 2 post inoculation, 50% working volume was changed daily with serum-free 199 medium for 4 days.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Mixing pancreatin and rotavirus G4 at a ratio of 10mg/L, incubating at 37deg.C for 1 hr, mixing pancreatin and rotavirusAdding the obtained mixture into a reactor to obtain a reactor in which the internal Rhabdoviral MOI is 0.001, continuously adding pancreatin at a rate of 200 μg/L/h to obtain a final concentration of pancreatin in the culture solution of Rhabdoviral of 8mg/L, and adding CaCl at a rate of 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (2) mmol/L. The cells were incubated at pH 7.2 at 100rpm and 37℃for 40 hours. At this time, the titer of rotavirus in the culture supernatant was about 8.2lg CCID 50 /mL。
EXAMPLE 4G 1 Rotavirus WAVE reactor culture
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 5L stirred bioreactor with 10 g/L3D TableTrix microcarriers, 5% serum MEM medium, 37℃at 80rpm, pH 7.2, dissolved oxygen 50% culture. The Cytodex I microcarriers grown on Vero cells were washed using MEM medium without serum.
2) Microcarriers grown on Vero cells were introduced into a culture bag at a rate of 8G/L, MEM medium having a final concentration of pancreatin of 5mg/L and a final concentration of calcium chloride of 2mM was added, and the G1-type rotavirus was inoculated at MOI=0.01, and the rotavirus was cultured at 37℃under aeration at pH 7.2 for 48 hours. Detection of rotavirus culture supernatant virus titres approximately 8.0lg CCID 50 /mL。
EXAMPLE 5 G1 rotavirus reactor culture and preparation of viral supernatant
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 10L basket style bioreactor (NBS Co.) with 10g/L Cytodex1 microcarriers and 8% serum 199 medium, and incubated at 38℃40rpm, pH7.4 with dissolved oxygen 35%. The microcarriers were grown for 5 days from day 2 after inoculation of the cells by sedimentation with 199 medium containing 2% serum and 75% working volume each day.
2) The medium was subjected to sedimentation and 4-time exchange with serum-free 199 medium containing 1mmol/L of calcium salt, at which time the theoretical serum residue was 0.0078%.
3) Based on the cell count results, rotavirus type G1, pancreatin and CaCl 2 The solution was mixed and added to the reactor. In the virus culture solution in the reactor, according to cellsAs a result of the counting, the G1 rotavirus MOI=0.01, the final concentration of pancreatin is 1.5mg/L, caCl 2 The final concentration was 1mmol/L, and the culture was carried out at pH 7.2, 60rpm and 37℃for 60 hours. During this period, the sterile pancreatin solution was continuously fed using peristaltic pumps at a flow rate of about 200. Mu.g/L/h. The titer of the supernatant of the virus culture was about 6.2lg CCID 50 /mL。
EXAMPLE 6 G1 rotavirus reactor culture and preparation of viral supernatant
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 10L basket style bioreactor (NBS Co.) with 10g/L Cytodex1 microcarriers and 8% serum 199 medium, and incubated at 38℃40rpm, pH7.4 with dissolved oxygen 35%. The microcarriers were grown for 5 days from day 2 after inoculation of the cells by sedimentation with 199 medium containing 2% serum and 75% working volume each day.
2) The medium was subjected to sedimentation and 4-time exchange with serum-free 199 medium containing 1mmol/L of calcium salt, at which time the theoretical serum residue was 0.0078%.
3) Based on the cell count results, rotavirus type G1, pancreatin and CaCl 2 The solution was mixed and added to the reactor. In the virus culture solution in the reactor, according to the cell count result, G1 type rotavirus MOI=0.01, final concentration of pancreatin 2mg/L, caCl 2 The final concentration was 0.5mmol/L, and the culture was carried out at pH 7.2, 60rpm and 37℃for 60 hours. During this period, the sterile pancreatin solution was continuously fed using peristaltic pumps at a flow rate of about 200. Mu.g/L/h. The titer of the supernatant of the virus culture was about 7.2lg CCID 50 /mL。
EXAMPLE 7 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 5L stirred bioreactor with 1 g/L3D TableTrix microcarriers, 5% serum 199 medium, 37℃at 80rpm, pH 7.2, dissolved oxygen 50% culture. From day 2 post inoculation, 50% working volume was changed daily with serum-free 199 medium for 4 days.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Adding pancreatin 10mg/L and rotavirus G4 into the reactor respectively to make MOI of rotavirus in the reactor be 0.01, continuously adding pancreatin at 400 μg/L/h to make final concentration of pancreatin in rotavirus culture solution be 8mg/L, adding CaCl at 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (2) mmol/L. The cells were incubated at pH 7.2 at 100rpm and 37℃for 40 hours. At this time, the titer of rotavirus in the culture supernatant was about 8.2lg CCID 50 /mL。
EXAMPLE 8 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 5L stirred bioreactor with 1g/L SoloHill microcarriers and 5% serum 199 medium, 37℃at 80rpm, pH 7.2, dissolved oxygen 50% culture. From day 2 post inoculation, 50% working volume was changed daily with serum-free 199 medium for 4 days.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Adding pancreatin 10mg/L and rotavirus G4 into the reactor respectively to make MOI of rotavirus in the reactor be 0.01, continuously adding pancreatin at 400 μg/L/h to make final concentration of pancreatin in rotavirus culture solution be 8mg/L, adding CaCl at 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (2) mmol/L. The cells were incubated at pH 7.2 at 100rpm and 37℃for 40 hours. At this time, the titer of rotavirus in the culture supernatant was about 8.0lg CCID 50 /mL。
EXAMPLE 9 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density Vero cells were seeded into 5L stirred bioreactor with 10g/L SoloHill microcarriers and 5% serum 199 medium, 37℃at 80rpm, pH 7.2, dissolved oxygen 50% culture. From day 2 post inoculation, serum-free 199 medium was used daily to exchange 50% of the working volume.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Adding pancreatin 10mg/L and rotavirus G4 into the reactor respectively to make MOI of rotavirus in the reactor be 0.001, continuously adding pancreatin at 400 μg/L/h to make final concentration of pancreatin in rotavirus culture solution be 8mg/L, adding CaCl at 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (2) mmol/L. The cells were incubated at pH 7.2 at 100rpm and 37℃for 40 hours. At this time, the titer of rotavirus in the culture supernatant was about 8.1lg CCID 50 /mL。
EXAMPLE 10 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density MA-104 cells were seeded into 5L stirred bioreactor with 10g/L SoloHill microcarriers and 5% serum 199 medium, cultured at 37℃at 80rpm, pH 7.2, dissolved oxygen 50%. From day 2 post inoculation, serum-free 199 medium was used daily to exchange 50% of the working volume.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Adding pancreatin 10mg/L and rotavirus G4 into the reactor respectively to make MOI of rotavirus in the reactor be 0.001, continuously adding pancreatin at a rate of 200 μg/L/h to make final concentration of pancreatin in rotavirus culture solution be 12mg/L, adding CaCl at a rate of 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (2) mmol/L. Culturing at pH 7.2, 100rpm and 37deg.C, and when culturing for 20 hr, the complete shedding virus titer of cells is 5.2lg CCID 50 /mL。
EXAMPLE 11 G4 rotavirus culture
1) According to 4X 10 5 cell/mL seeding Density MA-104 cells were seeded into 5L stirred bioreactor with 10g/L SoloHill microcarriers and 5% serum 199 medium, cultured at 37℃at 80rpm, pH 7.2, dissolved oxygen 50%. From day 2 post inoculation, serum-free 199 medium was used daily to exchange 50% of the working volume.
2) After 3 times of sedimentation and liquid exchange of the 5 th day liquid exchange by using the serum-free 199 culture medium, the theoretical serum residual quantity is 0.0024 percent.
3) Adding pancreatin 10mg/L and rotavirus G4 into the reactor respectively to make MOI of rotavirus in the reactor be 0.001, continuously adding pancreatin at a rate of 200 μg/L/h to make final concentration of pancreatin in rotavirus culture solution be 4mg/L, adding CaCl at a rate of 0.6 mM/day 2 . CaCl in serum-free 199 Medium 2 The final concentration of (C) was 0.1mol/L. Adding CaCl 2 The culture solution was turbid, cultured at pH 7.2, 100rpm and 37℃and when cultured for 12 hours, the cells were completely detached and the titer of the agglomerated virus was 4.8lg CCID 50 /mL。
Example 12 Effect of homogenization temperature on viral titre
The supernatant of the rotavirus culture of example 1 was split into 2L/serving portions. And (3) regulating the temperature of cooling liquid of a homogenizer to 25 ℃, 15 ℃, 5 ℃ and-5 ℃ respectively, carrying out homogenizing temperature test on a part of split-packed rotavirus liquid after the temperature is stable, homogenizing the liquid for 3 times with uniform homogenizing parameters of 400bar in the test process, and detecting the virus titer of the last homogenized product (figure 5). The results show that the virus is more stable when homogenization is performed under low temperature conditions. The drop of rotavirus titer is not obvious under the test condition of-5-15 ℃, and the loss of rotavirus is smaller.
Example 13 Effect of different stabilizers on rotavirus stability
The vaccine protectant is prepared according to the following formula:
formula A comprises 60% of sucrose, 0.2mol/L of potassium phosphate salt and 0.4mol/L of sodium succinate, and has pH of 6.4;
formula B comprises 50% of sucrose, 0.4mol/L of potassium phosphate salt and 0.2mol/L of sodium succinate, and has pH of 6.4;
formula C:80% sucrose, 0.2mol/L potassium phosphate salt, 0.4mol/L sodium citrate, pH6.4;
formula D:70% sucrose, 0.2mol/L potassium phosphate salt, 0.2mol/L sodium citrate, 0.2mol/L sodium succinate, pH6.4.
The protectants of formulas a-D were mixed with the rotavirus solution prepared in example 2 at a ratio of 1:1, respectively, to prepare rotavirus immunogenic compositions, and 2mL of the rotavirus immunogenic composition was placed in 50mL of hydrochloric acid, and rotavirus titers were determined by sampling at different times to evaluate the acid-resistant effect of the protectants (fig. 6A). The above rotavirus immunogenic mixture was placed in an incubator at 37 ℃ for 7 days and tested for rotavirus titer changes (fig. 6B). The results in fig. 6 demonstrate that each of the four formulations a-D improved the acid resistance and thermal stability of rotavirus compared to the control group of sterile water.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Claims (10)

1. A method of producing a rotavirus vaccine, the method comprising:
inoculating rotavirus into a cell culture solution, wherein the cell culture solution contains cells loaded on microcarriers;
in the process of maintaining the concentration of pancreatin at 2-10 mg/L and Ca 2+ At a concentration of 1mM or more, to collect a rotavirus-containing supernatant.
2. The method according to claim 1, wherein the concentration of pancreatin is maintained in the range of 2-8 mg/L;
preferably, the Ca 2+ The concentration of (2) is maintained in the range of 1-2 mMAnd is enclosed inside.
3. The method according to claim 1 or 2, characterized in that by supplementing pancreatin and/or Ca 2+ Maintains pancreatin and/or Ca 2+ Is a concentration of (3).
4. The method according to claim 1 or 2, wherein the serum concentration of the cell culture broth is less than or equal to 0.05%, more preferably the cell culture broth is serum free;
preferably, the cell culture fluid is selected from 199 medium, MEM medium, DMEM medium, and M199 medium.
5. The method according to claim 1, wherein the rotavirus has a multiplicity of infection MOI of 0.001 to 0.1;
preferably, the rotavirus is selected from the group consisting of a G9 rotavirus, a G1 rotavirus, a G2 rotavirus, a G3 rotavirus, a G4 rotavirus and a P1A 8 rotavirus.
6. The method of claim 1, wherein the microcarrier is used in an amount of 1-10 g/L;
preferably, the microcarrier is selected from the group consisting of porous structures based on dextran, collagen, plastic, gelatin and cellulose;
preferably, the microcarrier is selected from the group consisting of CytodexCytpdex />3DAnd Cytopore->
7. The method of claim 1, wherein the cells are selected from Vero cells, MA-104 cells, MRC5 cells, GBK cells, 293 cells, BHK cells, and CHO cells.
8. The method of claim 1, further comprising homogenizing and filtering the supernatant to obtain rotavirus liquid;
preferably, the homogenizing temperature is-5-15 ℃ and the pressure is 50-500 bar;
preferably, the aperture of the filter membrane used for the microfiltration is 0.2-1 μm;
preferably, the method does not include concentrating the supernatant.
9. The method of claim 8, wherein the method further comprises: mixing the rotavirus liquid with a protective agent;
preferably, the protectant comprises 20 to 60% saccharide, 0.05 to 0.5mol/L phosphate, and 0.05 to 0.5mol/L carboxylate;
more preferably, the saccharide is selected from sucrose, lactose, dextran, trehalose, galactose, sorbitol and mannose or a combination thereof;
more preferably, the carboxylate is selected from citrate and/or succinate;
more preferably, the ratio of the rotavirus liquid to the protective agent is 1:1-1:10.
10. A rotavirus vaccine prepared by the method of any one of claims 1 to 9.
CN202310698910.0A 2023-06-12 2023-06-12 Rotavirus vaccine and method for producing rotavirus vaccine Pending CN116836944A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070475A (en) * 2023-10-13 2023-11-17 金宇保灵生物药品有限公司 Bovine rotavirus serum-free suspension culture method, product and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117070475A (en) * 2023-10-13 2023-11-17 金宇保灵生物药品有限公司 Bovine rotavirus serum-free suspension culture method, product and application thereof
CN117070475B (en) * 2023-10-13 2023-12-29 金宇保灵生物药品有限公司 Bovine rotavirus serum-free suspension culture method, product and application thereof

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