JP5084214B2 - IPV vaccine - Google Patents

Description

  The present invention relates to a polio vaccine, particularly an inactivated polio vaccine containing an inactivated Sabin strain poliovirus (sIPV), and a method for producing the same.

  Polio is an infection caused by poliovirus. Poliovirus infects humans orally, grows in the intestinal tract, and enters the central nervous system via blood. Poliovirus that has entered the central nervous system grows in large motor neurons, causing motor neurons to degenerate and necrotic, causing acute flaccid paralysis in the extremities. In addition, if poliovirus invades the respiratory center of the medulla, it can lead to death from respiratory paralysis. Polio vaccines are widely used to suppress the onset of polio that causes such severe symptoms.

Two types of polio vaccines are used: oral live polio vaccine and inactivated polio vaccine. The oral live polio vaccine is a vaccine using an attenuated strain of poliovirus (Sabin strain). Orally administered attenuated strains of poliovirus cause normal infection. Polioviruses derived from oral live polio vaccines proliferate well in the human intestinal tract and consequently form immunity in the intestinal tract. Furthermore, production of antibodies in the blood is promoted by poliovirus derived from an oral live polio vaccine entering the blood and causing viremia. However, the ability of the attenuated strain of poliovirus to proliferate in the central nervous system is so weak that it usually does not cause paralysis. In addition, poliovirus derived from an oral live polio vaccine grows and is discharged from the stool for about 4 to 6 weeks after inoculation. This evacuated virus infects humans with weak or no immunity against polio around the recipient and exhibits an immunizing or enhancing effect as in the recipient.
However, the attenuated poliovirus derived from an oral live polio vaccine is mutated in the direction of virulent toxicity while it repeats growing in the recipient's body or in the human body infected with the excreted virus. It may happen. The mutant strain can cause vaccine-related paralysis very rarely.

  An inactivated polio vaccine is a vaccine in which poliovirus is inactivated by formalin to lose its infectivity. Inactivated polio vaccines will not cause vaccine-related paralysis because they will not grow in the recipient's body or infect humans around the recipient. In the production of inactivated polio vaccines, highly virulent strains have been used in the past, but in recent years development of attenuated strains (Sabin strains) has also been promoted (Non-Patent Document 1: Biologicals 34 (2006) 151-154). Non-patent document 2: Dev Bil. Basel. Karger, 2001, vol. 105, pp 163-169, Non-patent document 3: Clinical and virus Vol. 30 No. 5 (2002.12) 336-343). It was considered that the growth of the attenuated strain (Sabin strain) was slightly worse than that of the highly virulent strain.

Vero cells are passage cells derived from the kidneys of green monkeys and are widely used for virus culture because of their broad sensitivity to various viruses. There has been reported a method for producing an enterovirus 71 vaccine comprising culturing Vero cells on a microcarrier and propagating enterovirus 71 using the cultured Vero cells (Non-patent Document 4: Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development: Suh-Chin Wu, etc., Vaccine, 22, 3858-3864, 2004). General cell culture conditions using microcarriers have also been reported (Non-patent Document 5: Microcarrier cell culture principles & methods: Pharmacia LKB, Biotechnology, 1988).
Biologicals 34 (2006) 151-154 Dev Bil. Basel. Karger, 2001, vol. 105, pp 163-169 Clinical and virus Vol. 30 No. 5 (2002.12) 336-343 Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development: Suh-Chin Wu, etc., Vaccine, 22, 3858-3864, 2004 Microcarrier cell culture principles & methods: Pharmacia LKB, Biotechnology, 1988

  In the said situation, the manufacturing method of the inactivated polio vaccine containing the inactivated sabin strain poliovirus (sIPV) including the process of manufacturing the high titer Sabin strain poliovirus was desired.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors cultured Vero cells inoculated with Sabin strain poliovirus in the presence of about 4 g / L to about 6 g / L of microcarriers. It was found that a high-titer Sabin strain poliovirus can be produced. As a result of repeated studies based on these findings, the present invention has been completed.

That is, the present invention
(1) A method for producing a Sabin strain inactivated polio vaccine,
(A) culturing Vero cells inoculated with virus in the presence of about 4 g / L to about 6 g / L of microcarriers;
A method characterized by comprising:
(2) Furthermore,
(B) infecting the Vero cells with Sabin strain poliovirus;
(C) growing the poliovirus;
(D) recovering a virus solution containing the poliovirus, and (e) inactivating the poliovirus.
The method according to (1) above, comprising:
(3) The method according to (1) above, wherein the concentration of the microcarrier is about 5 g / L;
(4) The method according to (1) above, wherein the microcarrier is a dextran microcarrier;
(5) The method according to (1) above, wherein the step of growing Vero cells (step (a)) is performed on a scale of about 3 L or more;
(6) The method according to (1) above, wherein the step of growing Vero cells (step (a)) is performed on a scale of about 30 L or more;
(7) Furthermore,
(D-2) a step of purifying the virus solution,
The method according to (2) above, comprising:
(8) The purification step (step (d-2))
(I) pelletization of the virus solution collected in the step (d) by ultracentrifugation,
(Ii) sonication of the resuspended liquid of the pellet;
(Iii) purification by column chromatography,
The method according to (7) above, comprising:
(9) The method according to (8) above, wherein the purification by column chromatography (iii) is performed only once;
(10) Provided is a vaccine produced by the method described in (1) above.

  High-titer Sabin strain poliovirus can be obtained by culturing Vero cells inoculated with Sabin strain poliovirus in the presence of about 4 g / L to about 6 g / L microcarriers. By using a high titer Sabin strain poliovirus, an inactivated Sabin strain poliovirus can be efficiently produced. Therefore, a method for producing an inactivated polio vaccine (production method of the present invention) comprising a step of culturing Vero cells inoculated with Sabin strain poliovirus in the presence of about 4 g / L to about 6 g / L of microcarriers is not possible. The present invention is useful as an efficient method for producing an inactivated polio vaccine containing an activated Sabin strain poliovirus.

The present invention provides a method for producing an inactivated polio vaccine containing an inactivated sabin strain poliovirus (sIPV), comprising a step capable of producing a high titer Sabin strain poliovirus.
Hereinafter, the present invention will be described in detail.

1. Inactivated Sabin strain poliovirus (1) Sabin strain poliovirus In the present specification, “Sabin strain poliovirus” refers to Albert B. et al. It means a poliovirus strain derived from an attenuated strain of poliovirus isolated by Dr. Albert B. Sabin (Sabin AB, Boulger LR. History of Sabin attenuated poliovirus oral live vaccine strains. J. Biol. Standard 1973, 1, 115-118 etc.).
Sabin strain polioviruses include Sabin I strain poliovirus, Sabin II strain poliovirus and Sabin III strain poliovirus. Examples of the Sabin I type poliovirus include LSC, 2ab strain. Examples of the Sabin type II strain poliovirus include P712, Ch, 2ab strain and the like. Examples of the Sabin type III strain poliovirus include Leon, 12a ₁ b strain and the like.

(2) Inactivation In this specification, “inactivation” of virus means that the infectivity of virus is lost. Examples of inactivation methods include physical methods (for example, methods using X-ray irradiation, heat, ultrasonic waves, etc.), chemical methods (for example, methods using formalin, mercury, alcohol, chlorine, etc.) and the like. However, it is not limited to these.
Poliovirus can be inactivated by a known method (for example, see Biologicals 34 (2006) 151-154). Specifically, for example, poliovirus can be inactivated by treatment with formalin.

(3) Immunogenicity of inactivated Sabin strain poliovirus Inactivated Sabin strain poliovirus usually contains two viral antigens called D antigen and C antigen. D antigen is a complete viral particle. Antibodies against the D antigen have the ability to neutralize live virus infectivity and function as protective antibodies. The C antigen is called a defective particle, which is a particle obtained by multiplying a complete viral particle by a central nucleic acid RNA and a part of a viral protein, and is a hollow particle. Antibodies against the C antigen are very low if not capable of neutralizing the infectivity of live virus. Therefore, when using an inactivated Sabin strain poliovirus as a vaccine, the D antigen is required.
There are three types of poliovirus: type I, type II, and type III. Immunity against poliovirus infection is specific for the three virus types, type I, type II, and type III, with little if any cross-immunity between the types. Type I, type II and type III inactivated Sabin strain poliovirus D antigen is capable of producing neutralizing antibodies against type I, type II and type III wild-type (strongly virulent) polioviruses, respectively. have. D antigen of inactivated Sabin strain poliovirus has different immunogenicity in each of type I, type II and type III and neutralizes wild type (strongly virulent) poliovirus of type I, type II and type III The amount of D antigen required to produce sufficient antibodies to do so depends on the virus type.

  As described above, there are three types of poliovirus, type I, type II, and type III, and all types cause the same polio. Therefore, when inactivated Sabin strain poliovirus is used as a vaccine (inactivated polio vaccine), it is sufficient to neutralize poliovirus of wild strains (strongly virulent strains) of type I, type II and type III respectively. It is necessary to have an immunogenicity capable of producing a novel antibody, and it is preferable that the immunogenicity is close to the immunogenicity of an inactivated polio vaccine derived from a virulent strain conventionally used. In order to show such immunogenicity, the vaccine is in a ratio of 2 to 4:80 to 120: 80 to 120 in the inactivated Sabin strain poliovirus of type I, type II and type III in the amount of D antigen. In particular, it is preferably contained at a ratio of about 3: 100: 100. The inactivated Sabin strain poliovirus used in the present invention has an inactivated polio vaccine (eg, soak vaccine) derived from a virulent strain by having type I, type II and type III have the specific ratio as described above. Shows similar immunogenicity.

2. Production of inactivated Sabin strain poliovirus The inactivated Sabin strain poliovirus used in the present invention can be produced by the following method.
First, Vero cells are cultured in the presence of about 4 g / L to about 6 g / L of microcarriers to obtain poliovirus culture cells. The obtained poliovirus culture cells are inoculated with a seed virus (Sabin strain poliovirus) and cultured to obtain Sabin strain poliovirus. The obtained Sabin strain poliovirus is inactivated to obtain an inactivated Sabin strain poliovirus. Depending on the rape virus used (Sabin type I strain, Sabin type II strain or Sabin type III strain), the corresponding inactivated Sabin strain poliovirus can be obtained. The virus may be concentrated and / or purified before or after virus inactivation.

  As described above, inactivated polio vaccines must have immunogenicity capable of producing sufficient antibodies to neutralize wild strains (strongly virulent strains) of type I, type II and type III, respectively. It is. However, the inactivated Sabin strain poliovirus D antigen is different in type I, type II and type III in immunogenicity. Therefore, an inactivated polio vaccine having immunogenicity capable of producing sufficient antibodies to neutralize wild strains (strongly virulent strains) of type I, type II and type III, respectively, contains type I, type II And by adjusting the amount of inactivated poliovirus of type III.

(Vero cells)
Vero cells are passage cells derived from the kidneys of green monkeys (Cercopithecus aethiops) and are registered in the ATCC (American Type Culture Collection). Vero cells have a fibroblast-like morphology, have a broad sensitivity to various viruses, and are easily used for subculture and maintenance, and thus are widely used for virus culture. As Vero cells available from ATCC, for example, ATCC numbers CCL-81 and CRL-1587 are known.

(Microcarrier)
In the present specification, “microcarrier” means a carrier that allows cells to adhere to the surface and that can be cultured in a suspended state in a liquid medium. There are no particular restrictions on the material, shape, size, etc., so long as the carrier can attach cells to the surface and cultivate cells in a suspended state in a liquid medium.
Examples of the material for the microcarrier include dextran, gelatin, collagen, polystyrene, polyethylene, polyacrylamide, glass, and cellulose. As the material of the microcarrier, dextran is preferable.
Examples of the shape of the microcarrier include a spherical shape (bead) and a disk shape. The shape of the microcarrier is preferably spherical.
The size of the spherical microcarrier is, for example, about 0.01 to 1 mm, preferably about 0.05 to 0.5 mm, and more preferably about 0.1 to 0.3 mm.
The microcarrier may be porous.
Examples of the spherical microcarrier used in the present invention include Cytodex 1 (trade name), Cytodex 3 (trade name), Cytopore (trade name) (hereinafter, manufactured by GE Healthcare Bioscience). Examples of the disk-shaped microcarrier include Cytoline 1 (trade name) and Cytoline 2 (trade name) (hereinafter, manufactured by GE Healthcare Bioscience). Examples of the porous microcarrier include Cytopore (trade name), Cytoline 1 (trade name), Cytoline 2 (trade name) (hereinafter, manufactured by GE Healthcare Bioscience). The microcarrier used in the present invention is particularly preferably a spherical dextran microcarrier. As the spherical dextran microcarriers, Cytodex 1 (trade name), Cytodex 3 (trade name) and Cytopore (trade name) are preferable, and Cytodex 1 (trade name) and Cytodex 3 (trade name) are particularly preferable. 1 (trade name) is preferred.

(Vero cell culture)
In the present invention, Vero cells are grown by culturing in the presence of about 4 g / L to about 6 g / L microcarriers. The microcarrier concentration is preferably about 4.5 g / L to about 5.5 g / L, more preferably about 5 g / L.
The step of growing the above Vero cells is carried out on a scale of preferably 3 L or more, more preferably 30 L or more, and particularly preferably 150 L or more as a liquid volume. Further, the step of growing Vero cells is usually performed on a scale of 1000 L or less.
When culturing Vero cells in the presence of a microcarrier, examples of the medium include ME medium (Science, Vol. 122, 501 (1952)) containing about 5 to 20 vol% calf serum or fetal calf serum, DME medium [ Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950) ] Is used. The medium is preferably a DME medium, more preferably a DME medium containing calf serum, and particularly preferably a DME medium containing about 5 vol% calf serum. The medium is changed as needed during the cell culture period. The pH is preferably about 6-8, more preferably about 6.5-7.5, especially about 7. The culture is usually performed at about 35 ° C. to 40 ° C. for about 5 to 9 days, and aeration and agitation are added as necessary. The dissolved oxygen concentration (DO) during cell culture is preferably about 60 to 90%, more preferably about 70 to 80%, and particularly preferably about 75%.

(Poliovirus culture)
Poliovirus can be cultured by inoculating a cultured cell of Vero cells with a Sabin strain poliovirus (rapeseed virus) and culturing the poliovirus in the cell. Infected cells can be cultured in the same manner as the above-described culture of Vero cells. As a medium used for culturing poliovirus, a 199 medium is preferable, a 199 medium added with sodium bicarbonate is more preferable, and a 199 medium added with 0.3 w / v% sodium carbonate is particularly preferable.
The culture temperature during virus culture is preferably about 30 ° C to 38 ° C, more preferably about 32 ° C to 36 ° C, and particularly preferably about 33 ° C to 35 ° C. The period of virus culture is preferably about 1 to 5 days, more preferably about 2 to 4 days, and particularly preferably about 3 days. The virus culture can be terminated using the cytopathic effect by poliovirus (poliovirus-infected cells cause cell rounding and detachment from the microcarrier) as an index.
As a seed virus, Sabin strain poliovirus previously cultured using green monkey primary kidney cultured cells may be used.

(Recovery of virus solution containing poliovirus)
After completion of virus culture, the microcarrier is removed and a virus solution containing the poliovirus (sometimes referred to as a poliovirus solution) is collected.
The microcarriers can be removed using, for example, a Teflon mesh (Teflon is a registered trademark) (for example, a pore diameter of 120 μm). Since the poliovirus remains (attaches) to the microcarriers remaining on the mesh, the poliovirus may be recovered by washing with a virus culture solution or the like.
The obtained poliovirus solution may be filtered through a filtration membrane (for example, a 0.2 μm filtration membrane) to remove cell debris.

The poliovirus fluid may be concentrated and / or purified before or after inactivation.
Examples of the concentration method include ultrafiltration, ultracentrifugation, and dialysis. As a concentration method, an ultrafiltration method and an ultracentrifugation method are preferable, and an ultrafiltration method and an ultracentrifugation method are preferably performed. Further, after performing the ultrafiltration method, an ultracentrifugation method is preferably performed. .
As the ultrafiltration membrane used in the ultrafiltration method, an ultrafiltration membrane usually used for virus concentration can be used. The molecular weight cutoff of the ultrafiltration membrane is preferably about 100 kDa. As a material for the ultrafiltration membrane, polyethersulfone is preferable.
Concentration of poliovirus by ultracentrifugation can be performed, for example, by pelletizing a poliovirus solution by ultracentrifugation at 100,000 g for 4 hours at 4 ° C. The pellet may be resuspended in a phosphate buffer or the like. Agglomerates can also be loosened by sonicating the resuspended liquid of the pellets. The ultrasonic treatment can be performed using a commercially available apparatus such as INSONATOR MODEL 200M (KUBOTA). The conditions for the sonication are not limited as long as the aggregates are loosened, and can be appropriately set according to the container used for the sonication, the ultrasonic output, the concentration of the resuspended liquid, and the like. Examples of the ultrasonic treatment conditions include ultrasonic treatment at 200 W for 3 to 10 minutes. Even if such sonication is performed, the immunogenicity of Sabin strain poliovirus is not lost, and it can be suitably used as a poliovirus vaccine.

Examples of the purification method include a method utilizing physical properties such as the size, density and sedimentation coefficient of the object to be purified, a method utilizing a chemical or physicochemical reaction (such as adsorption / desorption), and the like. It is not limited. More specifically, examples of the purification method include density gradient centrifugation, filtration (including ultrafiltration), ion exchange column chromatography, affinity chromatography, gel filtration chromatography, and salting out. Can be mentioned. As the purification method, column chromatography is preferable, ion exchange column chromatography is more preferable, and DEAE-ion exchange column chromatography is particularly preferable. The number of purifications by column chromatography may be performed until the required purity is obtained, and is not particularly limited, but is preferably performed with a minimum number of steps in terms of production efficiency.
Concentration and purification are preferably performed by pelletizing poliovirus liquid by ultracentrifugation, sonication of the resuspended liquid of the pellet, and purification by column chromatography. Further, purification by column chromatography is preferably performed only once in terms of production efficiency. Poliovirus liquid is concentrated and purified efficiently and sufficiently by pelletizing poliovirus liquid by ultracentrifugation, sonication of the resuspended liquid of the pellet and purification by column chromatography only once. be able to.

(Inactivation of poliovirus)
Poliovirus can be inactivated by a commonly used method. More specifically, for example, poliovirus can be inactivated by adding an inactivating agent to the poliovirus solution and reacting the poliovirus with the inactivating agent. As the inactivating agent, formalin is preferable. The inactivation condition is not particularly limited as long as the poliovirus is inactivated. The period of inactivation treatment is usually about 2 to 4 times, preferably about 2.5 to 3.5 times the period in which inactivation of poliovirus is confirmed, in order to avoid the remaining of inactivated poliovirus. More preferably, it is about 3 times.
For example, when formalin is used as an inactivating agent, the amount added is preferably about 0.001% to 0.1% (w / v), more preferably about 0.005% to 0.05% (w / v). v), particularly preferably about 0.01% (w / v). The inactivation temperature is particularly preferably about 37 ° C. The inactivation period varies depending on the type of inactivation agent, the concentration of the inactivation agent, the inactivation temperature, and the like. For example, when about 0.01% (w / v) formalin is used as an inactivation agent and the inactivation temperature is about 37 ° C., the inactivation period is preferably about 8 to 16 days, more preferably about 10 days. -14 days, particularly preferably about 12 days. If about 0.01% (w / v) formalin is used and the inactivation temperature is about 37 ° C., Sabin strain poliovirus is usually inactivated by 4 days.

4). Inactivated polio vaccine The inactivated polio vaccine of the present invention contains an inactivated Sabin strain poliovirus. The inactivated Sabin strain poliovirus used in the present invention can be produced by mixing inactivated Sabin type I strain poliovirus, inactivated Sabin type II strain poliovirus, and inactivated Sabin type III strain poliovirus. . As described above, the inactivated Sabin strain poliovirus contains type I, type II and type III inactivated Sabin strain poliovirus in the amount of D antigen in a ratio of 2-4: 80-120: 80-120. In particular, it is preferably contained in a ratio of about 3: about 100: about 100.

The inactivated polio vaccine of the present invention can be produced and used according to conventional means. Specifically, it can be produced and used as described below.
The inactivated polio vaccine of the present invention can be formulated as an injection according to conventional means. The injection is prepared according to a method known per se, for example, by dissolving, suspending or emulsifying the above substance in a sterile aqueous or oily liquid usually used for injection. As the aqueous liquid for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants are used. The prepared injection solution is usually filled in an appropriate ampoule, syringe or the like.
The inactivated polio vaccine of the present invention may contain formulation additives such as preservatives, antioxidants, and chelating agents as necessary. Examples of preservatives include thimerosal and 2-phenoxyethanol. Examples of the chelating agent include ethylenediaminetetraacetic acid and glycol etherdiaminetetraacetic acid.
The inactivated polio vaccine of the present invention may further contain an adjuvant. Examples of the adjuvant include aluminum hydroxide, aluminum phosphate, and aluminum chloride.

  The inactivated polio vaccine of the present invention may further contain an immunogenic component other than the inactivated Sabin strain poliovirus. Examples of such immunogenic components include immunogenic components against viruses or fungi other than poliovirus. Examples of immunogenic components include toxoids, attenuated viruses, inactivated viruses, proteins, peptides, polysaccharides, lipopolysaccharides, lipopeptides, or combinations thereof. Examples of viruses or fungi other than poliovirus include pertussis, diphtheria, tetanus, influenza virus, measles virus, parotitis virus, rubella virus, herpes virus, chicken pox virus, rabies virus, human immunodeficiency Virus, hepatitis virus, Streptococcus pneumoniae, Neisseria meningitidis, Salmonella typhi, and influenza b.

The inactivated polio vaccine of the present invention can be administered parenterally, for example, by subcutaneous injection or intramuscular injection, preferably by subcutaneous injection.
The single dose of the inactivated polio vaccine of the present invention can be appropriately selected according to various conditions such as the age and weight of the administration subject. Specifically, for example, about 2 to 4 units (preferably about 3 units) using inactivated Sabin type I poliovirus as D antigen, and about 80 to 120 units (preferably using inactivated Sabin type II poliovirus as D antigen) About 100 units) and inactivated Sabin type III poliovirus as D antigen may contain about 80 to 120 units (preferably about 100 units).
The frequency of administration of the inactivated polio vaccine of the present invention may be administered 2 to 3 times at intervals of 3 to 8 weeks, for example, as the first immunization. As booster immunization, for example, it may be administered once more at an interval of 6 months or more after the initial immunization.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.

Reference Example 1 (Establishment of a preservation cell bank for polio vaccine virus production)
A stock cell bank for vaccine virus production was prepared from Vero cells obtained from ATCC by the following procedure.
(I) Preparation of Seed Cell Bank (MCB: Master Cell Bank) Thaw frozen ampoule cells (CCL 81 Vero, F-6573, passage number 124) received from ATCC and empty 4 ounce bottle (capacity 154 mL) And a culture bottle having a cell growth area of 54 cm ² . Cell growth solution (5vol% calf serum, 0.075% sodium bicarbonate, 20μg / mL erythromycin, 100μg / mL kanamycin (final concentration) added DME (Dulbecco's Modified Eagle's Medium, Sigma, Catalog No. D5523) ) Was added dropwise over about 5 minutes. The bottle containing the cells and cell growth solution was statically cultured at 36 ° C. (one 4-ounce bottle, 125 generations). The next morning, the cell growth solution was replaced with a new 15 mL, and the cells were further incubated at 36 ° C. Subculture was performed on the 6th day after the start of static culture (from one 4-ounce bottle to four 4-ounce bottles, 126 generations). The passage method was performed according to the following procedure.

Passage method
(1) Discard the culture solution.
(2) Place 5 mL of 0.25% trypsin solution for passage into a 4 ounce bottle.
(3) Soak the cell surface for about 1 minute and discard the passage 0.25% trypsin solution.
(4) Place the 4 ounce bottle at 36 ° C and wait for the cells to peel off the glass surface.
(5) When cells begin to detach, add 5 mL of cell growth solution and detach all cells by pipetting.
(6) Further float the cells uniformly by pipetting, and transfer the cell growth solution to a centrifuge tube.
(7) Centrifuge at 600 rpm for 5 minutes, discard the supernatant, and float the sedimented cells evenly in about 8 mL of new cell growth solution by pipetting.
(8) Add 2 mL of cell suspension to 4 new 4 ounce bottles (13 mL of new cell growth solution is dispensed).
(9) Place four 4-ounce bottles at 36 ° C and perform cell culture.

The composition of passage 0.25% trypsin solution was as follows.
5% trypsin ^{* 1} 50 mL / L
5% Polyvinyl pyrrolidone (90K) 20 mL / L
0.247 mol Sodium edetate ^{* 2} 56 mL / L
EK ^{* 3} 2 mL / L
Trypsin dilution ^{* 4} 872 mL / L

* 1: Trypsin having a 1: 300 activity derived from porcine pancreas was used.
* 2: The composition of 0.247 mol edetate sodium was as follows.
Sodium edetate-2Na ・ 2H ₂ O 91.95 g / L
NaOH 9.88 g / L
* 3: The composition of EK was as follows.
Erythromycin Lactobionate 10,000μg / mL
Kanamycin Sulfate 50,000μg / mL
* 4: The composition of the trypsin dilution was as follows.
NaCl 8,000 mg / L
KCl 400 mg / L
Na ₂ HPO ₄・ 12H ₂ O 150 mg / L
KH ₂ PO ₄ 60 mg / L

Subsequent passages are repeated at intervals of 3 to 6 days, with the same method (the culture area ratio is approximately 3 to 4 times per passage, so the amount of liquid handled differs from the culture bottle). (The number of passages is increased by one for each passage). In 33 SR bottles (Small Roux bottles: culture bottles with a capacity of 727 mL and a cell growth area of 156 cm ² ), the cultured cells in the 129th generation were trypsinized and centrifuged in the same way as at the passage, and the sediment was stored in a cryopreservation medium (10 About DME (Dulbecco's Modified Eagle's Medium, Sigma, Catalog No. D5523)) supplemented with% DMSO (Dimethyl Sulfoxide), 10vol% calf serum, 0.075% sodium bicarbonate, 20μg / mL erythromycin, 100μg / mL kanamycin (final concentration) Resuspended at 1.5 × 10 ⁷ cells / mL. 1 mL of the above cell suspension was dispensed into an ampoule, and the temperature was lowered to −32 ° C. with a slow freezer (decreasing by 1 ° C. in about 1 minute), then transferred to liquid nitrogen and stored. The 129th generation cells obtained as described above were used as a seed cell bank (MCB).

(Ii) Preparation of a production cell bank (MWCB) for manufacture Manufacturing the cell bank (MCB) of (i) above using the cell bank (MCB) prepared and stored in (i) above. The method was basically the same as the preparation method of No. 1, and cell growth from amputation through cell passage was performed up to generation 134 (because of the large number of starting cells, the amount of handling liquid, the type of culture bottle and the number were Different). The 134 cells were also stored in liquid nitrogen in the same manner as the seed cell bank (MCB). The 134th generation cells obtained as described above were used as a stock cell bank for manufacturing (MWCB).

Example 1 (Preparation of cells for producing polio vaccine virus)
(I) Stationary culture process One ampoule (cell of generation 134) of the preserved cell bank for manufacturing (MWCB) prepared and stored in the above Reference Example 1 was used for the seed cell bank (MCB) of the above Reference Example 1 and for manufacturing. Thaw the cells in the same way as in the preparation of the preserved cell bank (MWCB), and leave the cells for 7 days in 3 LR bottles (Large Roux bottles, culture bottles with a capacity of about 1540 mL and a cell growth area of about 274 cm ² ). Cultured (135s). On the 7th day after the start of static culture, subculture was performed, and the culture scale was expanded to 18 LR bottles (136th generation) to perform static culture. The stationary culture and its passage were performed in the same manner as the preparation of the seed cell bank (MCB) and the preparation of the storage cell bank for manufacturing (MWCB) in Reference Example 1 above.
Next, it was statically cultured for 7 days in a 40-stage cell factory (Nunk, catalog No. 139446) (137th generation). The cells were passaged on the seventh day after the start of static culture, and further statically cultured for 7 days in four 40-stage cell factories (generation 138).

(Ii) Microcarrier culture step Next, cells in the 138th generation obtained in the stationary culture step in (i) above were trypsinized and centrifuged in the same manner as in the subculture in (i) above, resulting in sediment. DME supplemented with 1,000 mL of microcarrier culture cell growth solution (5 vol% calf serum (Thermo Trace), 0.11% sodium bicarbonate, 0.1% fructose, 20 μg / mL erythromycin, 100 μg / mL kanamycin (final concentration)) (Dulbecco's Modified Eagle's Medium, Sigma, Catalog No. D5523)) was suspended evenly by pipetting. The cell suspension is swollen with PBS (Phosphate Buffered Saline) in advance and mixed with microcarriers (Cytodex 1 (trade name), GE Healthcare Bioscience) equilibrated with cell growth solution for microcarrier culture (Cytodex 1 ( (Trade name) was 5 g / L by weight before swelling), and cultured in 3 units of 50 L capacity incubator at 37 ° C. and pH 7.15. From the second day of culture, half of the cell growth liquid was continuously replaced with new cell growth liquid per day. Cells cultured for 7 days were used as polio vaccine virus production cells (139th generation).

Example 2 (Production of inactivated polio vaccine type I)
(I) Virus culturing step The polio vaccine virus-producing cells obtained in Example 1 (139th generation) were agitated immediately before inoculation with the seed virus to precipitate the cells, and 0.075% sodium bicarbonate, 20 μg / mL erythromycin. The plate was washed once with EBSS (Earl's Balanced Salt Solution) added with 100 μg / mL kanamycin (final concentration). After removing 5 mL of the cell culture supernatant collected with the microcarriers, add 0.25% trypsin solution to make 5 mL again, detach the cells from the beads, float them, count the number of cells, and count the total number of cells in the 50 L volume incubator. Estimated. Each cell was inoculated with an attenuated Sabin type I (LSc, 2ab strain) rape virus at a concentration of about 10 ⁻³ CCID ₅₀ . Immediately after rape virus inoculation, 50 L of a virus culture solution (0.3% sodium hydrogen carbonate, 20 μg / mL erythromycin, 100 μg / mL kanamycin (final concentration) added M199 (Medium 199)) was injected into the incubator. The seed virus used was cultured in advance in African green monkey primary kidney cultured cells at about 33.3 ° C., and then subdivided and stored frozen at −70 ° C.
Virus culture was performed at 34 ° C. ± 1 ° C. for 3 days. Using the cytopathic effect of poliovirus (poliovirus-infected cells cause cell rounding and then detached from the microcarrier) as an index, the virus culture was terminated when the cells were detached from the microcarrier by 95 to 100%. After completion of the virus culture, the microcarrier was removed through a Teflon mesh (Teflon is a registered trademark) (pore size: 120 μm), and the virus suspension was recovered. The microcarriers remaining on the mesh were washed once with about 3 L of virus culture medium per 50 L volume incubator. This washing solution was added to the collected virus suspension to obtain a “type I poliovirus solution”.

(Ii) Virus Concentration / Purification Step About 150 L of the type I poliovirus solution obtained in (i) above was filtered through a 0.2 μm filtration membrane (Pole, SLK7002NRP) to remove cell debris. The filtrate was concentrated to 1.2 L with an ultrafiltration membrane (Sartorius, PESU (Polyethersulfone) 100 kDa, 0.1 m ² 3051466801E--SG). The concentrated virus solution was pelleted by ultracentrifugation at 100,000g for 4 hours at 6 ° C, and resuspended in PB (Phosphate Buffer, 0.1 mol / L) (1 mL of centrifuge tube (about 100 mL) pellet was added to 5 mL. Resuspended in PB). The pellet resuspension was shaken overnight at 4 ° C. and then subjected to ultrasonic treatment (Kubota, INSONATOR MODEL 200M) at 200 W for 8 minutes to loosen the agglomerates. The supernatant was collected after centrifugation at 15,000 rpm for 30 minutes. The obtained supernatant was purified by DEAE Sepharose CL-6B (trade name, GE Healthcare Biosciences, GE 17-0710-05). PB (Phosphate Buffer, 0.1 mol / L) was used as an eluent. The first peak was collected by monitoring at an absorbance of 280 nm to obtain a “purified type I poliovirus solution”. For the collected peak, 260/280 nm was calculated as the absorbance value, and it was confirmed that it was 1.5 or more (260/280 nm of poliovirus complete particles is 1.6 to 1.7).

(Iii) Inactivation step The purified type I poliovirus solution obtained in (ii) above is diluted with a pre-inactivation dilution solution (containing 5% aminoacetic acid (final concentration), including Ca, Mg, Phenol Red, and sodium bicarbonate. Diluted about 10 times with M199) and filtered through a 0.2 μm filter membrane (Pall, SLK7002NRP) to remove virus clumps. After preparing the filtrate, inactivation was started immediately so that the virus would not aggregate again. One hour before the start of inactivation, the filtrate and 1: 200 diluted formalin were separately heated at 37 ° C. While thoroughly stirring the filtrate, formalin was added to a final concentration of 1: 4,000, and the mixture was heated to 37 ° C. to initiate inactivation. During formalin treatment, the inactivation of the virus was promoted uniformly by stirring the solution twice daily in the morning and afternoon. Assuming that a virus with insufficient inactivation attaches to a container stopper or a specific place on the container, replace the stopper on the second and fourth days of inactivation, and replace the container on the sixth day. Went. In consideration of the possibility of virus aggregation during the inactivation step, filtration was performed with a 0.2 μm filter membrane (Pall, SLK7002NRP) on the 6th day of inactivation. The formalin treatment process was completed in 12 days. On the 12th day, formalin-treated virus solution free formalin was neutralized with sodium sulfite (added to 0.0264 mol / L), and sodium edetate was added as a stabilizer (0.0009 mol / L). A stock solution of “Vaccine Type I” was used.

(Iv) The amount of D antigen is measured by an indirect ELISA method using an antibody having high specificity for the type and D antigen. In the indirect ELISA method, first, a D-antigen-specific monoclonal antibody (derived from a mouse) of the same type as the test antigen is coated on a microplate as a primary antibody. Next, the test antigen is diluted and placed. Next, a rabbit polyclonal antibody of the same type as the test antigen is loaded as a secondary antibody, and further a HRPO-labeled anti-rabbit IgG antibody is loaded and reacted. After the reaction, the color is developed using an orthophenylenediamine solution, and then the absorbance at 492 nm is measured. The D antigen amount of the test antigen is determined by comparing the measured value of the absorbance of the test antibody with the measured value of the control antigen by the parallel line quantification method.

Example 3 (Production of inactivated polio vaccine type II)
As in Example 2, except that attenuated Sabin strain type II (P712, Ch, 2ab strain) was used instead of attenuated Sabin strain type I (LSc, 2ab strain) A stock solution of “Polio vaccine type II” was produced.

Example 4 (Production of inactivated polio vaccine type III)
As seed virus, I-type attenuated Sabin strains (LSc, 2ab strains) III-type attenuated Sabin strains in place of (Leon, 12a ₁ b strain) other was used, in the same manner as in Example 2, "inactivated A stock solution of “Polio vaccine type III” was produced.

Example 5 (Production of inactivated polio vaccine)
The stock solutions of inactivated polio vaccine type I, inactivated polio vaccine type II and inactivated polio vaccine type III obtained in Examples 2, 3 and 4 above were adjusted so that the amount of D antigen was 3, 100 and 100, respectively. 199 medium (M199) was prepared, and 2-phenoxyethanol was mixed to a final concentration of 0.5 vol%. The obtained mixed solution was adjusted to pH 7 with sodium hydroxide or hydrochloric acid to obtain an inactivated polio vaccine.

Example 6 (culture of Vero cells and poliovirus by microcarrier method)
(I) Vero cell culture
After starting from the Vero cell working bank (MWCB93), the cells subcultured by static culture were detached using trypsin-EDTA solution (0.25% trypsin, 0.014M EDTA), and centrifuged at 600 rpm for 10 minutes. The suspension was suspended in a growth medium (5 vol% calf serum, 0.11% sodium hydrogen carbonate (NaHCO ₃ ), 0.1% fructose, 20 μg / mL erythromycin and 100 μg / mL kanamycin-added Dulbecco's modified Eagle Medium (DME)).
A microcarrier (Cytodex 1 (trade name)) was swelled with PBS (−), autoclaved at 121 ° C. for 15 minutes, and replaced with a cell growth medium.
The culture device uses New Brunswick Scientific's Serigen Plus and Serigen. After adding microcarriers and cell suspension, the cell growth medium is adjusted to a final cell growth rate of 4.8 L (3.5 L for Serigen), and the culture temperature is 37.0. C., dissolved oxygen concentration (DO): 15%, PH: 7.15, rotation speed: 35-50 rpm. The medium was replaced with a cell growth medium with a NaHCO ₃ concentration of 0.15% as the replacement medium, and the amount of about 4 L / day was continuously applied from the second day of culture (in the case of serigen, the total amount was changed every other day). ). The number of cells was measured using a Coulter counter (trade name).
The results of cell culture are shown in FIG. 1 (serigen was used only for the 3H experiment). With carrier 3g / L, low concentration of starting cells (3L, approx. 2 × 10 ⁵ cells / mL) on the 7th day, high concentration of starting cells (3H, approx. 10 × 10 ⁵ cells / mL), 8 days The number of cells in the eye was maximal and then declined. In addition, in the carrier 5g / L, the cell number increased for 11 days at the low concentration of starting cells (5L, approximately 2 × 10 ⁵ cells / mL), but the cell number decreased on the 12th day. In the case of a low concentration of starting cells, the total cell number of carrier 5 g / L was superior on day 10 compared to 3 g / L, but the difference was not significant. High concentration of the starting cell number in carriers 5 g / L in (5H, about 10 × 10 ⁵ cells / mL) in 9 days is still in increasing stage of cell number, cell number at this point was about 2.8 × 10 ⁶ cells / mL, and grew to about 2.7 times the number of starting cells.

(Ii) Culture of poliovirus Type 1 poliovirus (IS-90C) was used as the seed virus. On the final day of cell counting, the cells were washed with EBSS supplemented with 0.075% NaHCO ₃ , 4 times the culture volume, and then M-199 (E) medium supplemented with 0.3% NaHCO ₃ , 20 μg / mL erythromycin and 100 μg / mL kanamycin ( Virus culture medium) 1 L of seed virus was prepared by dilution. This was inoculated into the washed cells, and the medium for virus culture was added to the culture volume of each culture apparatus. Virus culture was performed at a culture temperature of 33.3 ° C., DO: 15%, pH: 7.40, and rotation speed: 35 to 50 rpm. When the cells completely detached from the microcarrier due to viral CPE (cytopathic effect), the virus culture was stopped and the virus solution was harvested. The harvested virus solution was stored frozen at -80 ° C.
In this test, virus culture was started on the last measurement day of each cell growth curve shown in FIG.
The virus titer was measured as follows. GMK-2 cells cultured for 3 days in a roller tube were washed twice with 0.075% NaHCO ₃ , 200u / mL penicillin, 200μg / mL streptomycin and 1 mL of HBSS, followed by cell maintenance solution (0.1% bovine serum albumin, 0.225% NaHCO _3. 200 mL / mL penicillin and 200 μg / mL streptomycin added M-199 medium) were added. The test virus solution was diluted 0.5 log ₁₀ steps with the cell maintenance solution, and 10 ⁻⁷ to 10 ^−8.5 virus solution was inoculated into 0.2 mL for each tube and 5 tubes for each dilution. After inoculation, the cells were cultured in a 36 ° C. furan room for 7 days, and the infectivity titer (CCID ₅₀ /0.2 mL) was calculated by the Reed & Muench method based on the determination of CPE observation on the 7th day.
The infectivity titer (virus titer) of type I poliovirus obtained in Vero cells cultured under each condition is shown in FIG. As a result of the virus culture, the highest infectious titer was obtained in the high-concentration starting cell number system (5H) of carrier 5 g / L, which had a cell density of about 2.8 × 10 ⁶ cells / mL on the 9th day. Infectious titers were in the order of 5H, 5L, and 3L, which was consistent with the total cell count. In addition, the infectivity titer more than 10 times was obtained in the 5H system compared with the virus solution obtained by culturing in green monkey kidney cells as a control.

  High-titer Sabin strain poliovirus can be obtained by culturing Vero cells inoculated with Sabin strain poliovirus in the presence of about 4 g / L to about 6 g / L microcarriers. By using a high titer Sabin strain poliovirus, an inactivated Sabin strain poliovirus can be efficiently produced. Therefore, a method for producing an inactivated polio vaccine (production method of the present invention) comprising a step of culturing Vero cells inoculated with Sabin strain poliovirus in the presence of about 4 g / L to about 6 g / L of microcarriers is not possible. The present invention is useful as an efficient method for producing an inactivated polio vaccine containing an activated Sabin strain poliovirus. Moreover, since the inactivated polio vaccine of this invention can suppress the onset of polio effectively, it is useful as a vaccine with respect to polio.

FIG. 1 is a graph showing a growth curve of Vero cells in the culture of Vero cells by the microcarrier method. 3L indicates 3 g / L of microcarrier with a starting cell number of about 2 × 10 ⁵ cells / mL (low concentration). 3H indicates 3 g / L of microcarrier with a starting cell number of about 10 × 10 ⁵ cells / mL (high concentration). 5 L indicates 5 g / L of microcarrier with a starting cell number of about 2 × 10 ⁵ cells / mL (low concentration). 5H indicates 5 g / L of microcarrier with a starting cell number of about 10 × 10 ⁵ cells / mL (high concentration). FIG. 2 is a graph showing the infectivity titer (virus titer) of type I poliovirus obtained in Vero cells cultured under various conditions. 3L shows type I poliovirus obtained with Vero cells cultured with microcarriers 3 g / L at a starting cell number of about 2 × 10 ⁵ cells / mL (low concentration). 5L shows type I poliovirus obtained from Vero cells cultured at a microcarrier of 5 g / L with a starting cell count of about 2 × 10 ⁵ cells / mL (low concentration). 5H represents type I poliovirus obtained from Vero cells cultured at a microcarrier of 5 g / L with a starting cell number of about 10 × 10 ⁵ cells / mL (high concentration). Ref. Indicates type I poliovirus grown using green monkey kidney cells.

Claims (9)

A method for producing a Sabin strain inactivated polio vaccine comprising:
(A) culturing Vero cells inoculated with virus in the presence of 4 g / L to 6 g / L of microcarriers;
(B) infecting said Vero cells with a type I, II or III Sabin strain poliovirus;
(C) growing the poliovirus;
(D) a step of recovering a virus solution containing the poliovirus;
(E) inactivating the poliovirus; and
(F) Inactivated Sabin strain poliovirus of type I, type II and type III were mixed, and the ratio of D antigen amount (type I: type II: type III) of each inactivated sabin strain poliovirus was 2-4 : 80-120: the process of 80-120,
A method comprising the steps of: The method of claim 1, wherein the concentration of the microcarrier is 5 g / L.   The method according to claim 1, wherein the microcarrier is a dextran microcarrier. The method according to claim 1, wherein the step of growing Vero cells (step (a)) is performed on a scale of 3 L or more. The method according to claim 1, wherein the step of growing Vero cells (step (a)) is performed on a scale of 30 L or more. further,
(D-2) a step of purifying the virus solution,
Including method of claim 1, wherein. The purification step (step (d-2))
(I) pelletization of the virus solution collected in the step (d) by ultracentrifugation,
(Ii) sonication of the resuspended liquid of the pellet;
(Iii) purification by column chromatography,
The method of claim 6 , comprising: 8. The method according to claim 7 , wherein the purification by (iii) column chromatography is performed only once.   A vaccine produced by the method according to claim 1.

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