CN112195149B - Method for preparing rabies vaccine for human - Google Patents
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- CN112195149B CN112195149B CN202011130043.3A CN202011130043A CN112195149B CN 112195149 B CN112195149 B CN 112195149B CN 202011130043 A CN202011130043 A CN 202011130043A CN 112195149 B CN112195149 B CN 112195149B
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Abstract
The invention discloses a method for preparing a human rabies vaccine, and belongs to the field of vaccines. The method mainly comprises the steps of cell culture, virus inoculation, virus recovery, inactivation, purification, stock solution preparation and the like; the formula of the culture medium used for cell culture is as follows: 17.6g/L, GlutaMAX 2-4 mmol/L of VP-SFM, 1.5-2.0 g/L, Tricine 1.5-2.0 g/L of fructose, and 2.0-3.0 g/L of sodium bicarbonate is added to the cell maintenance solution used after virus inoculation on the basis of the culture medium. The method of the invention does not add human serum albumin and serum into the culture medium and the cell maintenance liquid, has higher safety and has production efficiency far higher than the industrial level.
Description
Technical Field
The invention belongs to the field of vaccines, and particularly relates to a method for preparing a human rabies vaccine.
Background
Rabies is a zoonosis caused by rabies virus, once the disease occurs, the mortality rate is 100 percent, and the damage is great. The prevention of rabies is still a serious public problem, and due to the long onset latency of rabies, the injection of rabies vaccine is the first choice for preventing rabies. The production of high-quality rabies vaccine for human use is a common pursuit goal of researchers in various countries.
However, in the existing production process of rabies virus vaccines on the market, in order to keep the virus titer higher and ensure the higher production efficiency, a culture medium containing serum or human serum albumin is used as a maintenance solution to protect the virus from the action of certain hydrolytic enzymes. However, the serum or human serum albumin increases the risk of contamination of the vaccine product containing animal-derived components, and meanwhile, the preparation process of the virus liquid containing human serum albumin may cause that beta-propiolactone changes the structure of human serum albumin to cause sensitization reaction during virus inactivation, thereby increasing the safety risk of products and increasing the production cost.
Disclosure of Invention
The invention aims to solve the problems that: provides a method for preparing the human rabies vaccine with production efficiency and vaccine safety considered.
The technical scheme of the invention is as follows:
a serum-free medium for preparing human rabies vaccine comprises the following formula:
VP-SFM is a commercially available (Gibco under Saimer Feishale)TM) Serum-free ultra-low protein (5 μ g/ml) medium, free of proteins, peptides or other animal or human derived components; designed for culturing VERO cells to produce virus. This medium is suitable for the cultivation of COS-7, MDCK, BHK-21 (suspension culture) and HEp2 cells, and for the production of recombinant proteins.
The serum-free culture medium is as described above, and the concentration of the GlutaMax is 4 mmol/L;
and/or the fructose concentration is 2.0 g/L;
and/or the concentration of Tricine is 2.0 g/L.
A cell maintenance solution for preparing rabies vaccine for human, which is prepared by adding sodium bicarbonate with final concentration of 2.0-3.0 g/L on the basis of the serum-free culture medium of claim 1 or 2.
The concentration of sodium hydrogencarbonate in the cell-maintaining solution was 3.0g/L as described above.
A method for preparing a rabies vaccine for human use, comprising the steps of:
(1) cell culture: culturing Vero cells to 1-1.5 x 10 in a sheet-shaped carrier bioreactor7After cells/ml, performing perfusion culture by using the serum-free culture solution;
(2) virus inoculation: inoculating rabies virus rPV-2061 into cells according to 1/400-1/100 MOI, inoculating for 6-8 h, and performing perfusion culture by using the cell maintenance liquid;
(3) and (3) toxin collection: harvesting the virus from 2-3 days after virus inoculation to obtain a virus harvesting solution;
(4) inactivation: inactivating the virus using beta-propiolactone;
(5) and (3) purification: purifying the virus by molecular sieve chromatography;
(6) preparing stock solution: adding protective agent to prepare stock solution.
The term "protective agent" refers to a substance that protects the biologically active substance from damage during lyophilization during vaccine production and is therefore also referred to as a "lyoprotectant".
As described above, the seed is rPV-2061 strain, PV-2061 strain, CTN strain or aG strain.
As in the method, the temperature of perfusion culture in the steps (1) and (2) is 33-35 ℃, the pH is 7.50 +/-0.1, the dissolved oxygen is 60 +/-20%, and the rotating speed is 90-110 rpm.
The method comprises the steps of (3) and (4) concentrating the virus harvest liquid by using an ultrafiltration membrane package, wherein the concentration multiple is preferably 20-30 times;
and/or the purified filler of the molecular sieve is agarose gel 4 FF.
The method as described above, further comprising a lyophilization step after step (6).
The rabies vaccine for human use is prepared by the method.
The invention has the following beneficial effects:
the culture medium and the cell maintenance liquid do not contain human serum albumin and serum, so that the safety of vaccine products is enhanced; on the other hand, the propagation of Vero cells and rabies viruses can be promoted, and the production efficiency of the human rabies vaccine is improved.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
Example 1 preparation of rabies vaccine of the present invention
1. Production cell
The production cells are Vero cells adapted to serum-free culture.
2. Poison seed
The seed virus for production is rabies virus rPV-2061 strain.
3. Stock preparation
(1) Cell culture: culturing 1 or more cells in a recovery working cell bank (P140) at 37 +/-1 ℃, amplifying and passaging to (P146) according to the ratio of 1: 3-1: 6, inoculating to a sheet-shaped carrier bioreactor (P147), wherein the inoculation density of the cells in the bioreactor is 3-5 multiplied by 105cells/ml. The formula of the culture medium for culturing the cells is as follows:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(2) Virus inoculation: when the cell density in the bioreactor reaches 1-1.5 × 107cell/ml, inoculating rabies virus (MOl: 1/400-1/100), culturing for 6-8 h after virus infection, and beginning perfusion of the cell maintenance solution.
The maintenance liquid formula comprises:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(3) And (3) toxin collection: perfusing and culturing until 2-3 days after the cells are infected with the virus, beginning perfusing to harvest virus liquid, wherein the droplet size of the harvested virus liquid is not less than 6.5LgLD50And/ml. Coarse-filtering with 0.65 μm filter element, passing through 300-500 kD membrane packageConcentrating by 20-30 times.
(4) Inactivation: adding beta-propiolactone into the concentrated virus harvest liquid according to the volume ratio of 1: 4000, and inactivating the rabies virus at 4 ℃ for 24 +/-2 hours; standing at 37 +/-1 ℃ for 2-3 hours to hydrolyze the beta-propiolactone.
(5) And (3) purification: selecting agarose 4FF as a filler to carry out molecular sieve chromatography purification, taking a phosphate buffer solution with pH of 7.2-8.0 as a mobile phase, wherein the loading amount is 3-8% of the column volume; the detector is an ultraviolet detector, the detection wavelength is 280nm, and the first chromatographic peak is the viral protein chromatographic peak.
(6) Preparing stock solution: adding protective agent to prepare stock solution.
4. Preparation of finished product
Adding protective agent to obtain semi-finished product, packaging, and lyophilizing to obtain the final product.
The volume of the protective agent depends on the content of the antigen in the stock solution, the volume of the freeze-drying protective agent needs to be adjusted, and the protein content is not higher than 80 mu g/dose (excluding the content of human serum albumin); 1mL of each dose.
Example 2 preparation of rabies vaccine of the invention
1. Production cell
The production cells are Vero cells adapted to serum-free culture.
2. Poison seed
The virus seed for production is rabies virus CTN strain.
3. Stock preparation
(1) Cell culture: culturing 1 or more recovered cells in a recovery working cell bank (P140) at 37 +/-1 ℃, amplifying and passaging to (P146) according to the ratio of 1: 3-1: 6, inoculating to a sheet-shaped carrier bioreactor (P147), wherein the inoculation density of the cells in the bioreactor is 3-5 multiplied by 105cells/ml. The formula of the culture medium for culturing the cells is as follows:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(2) Virus inoculation: when the cell density in the bioreactor reaches 1-1.5 × 107cell/ml, inoculating rabies virus (MOl: 1/400-1/100), culturing for 6-8 h after virus infection, and beginning perfusion of the cell maintenance solution.
The maintenance liquid comprises the following components:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(3) And (3) toxin collection: perfusing and culturing until 2-3 days after the cells are infected with the virus, beginning perfusing to harvest virus liquid, wherein the droplet size of the harvested virus liquid is not less than 6.5LgLD50And/ml. Coarse-filtering with a 0.65 μm filter element, and concentrating by 20-30 times with a 300-500 kD membrane module.
(4) Inactivation: adding beta-propiolactone into the concentrated virus harvest liquid according to the volume ratio of 1: 4000, and inactivating the rabies virus at the temperature of 2 ℃ for 24 +/-2 hours; standing at 37 +/-1 ℃ for 2-3 hours to hydrolyze the beta-propiolactone.
(5) And (3) purification: selecting agarose 4FF as a filler to carry out molecular sieve chromatography purification, taking a phosphate buffer solution with pH of 7.4-7.8 as a mobile phase, wherein the loading amount is 3-8% of the column volume; the detector is an ultraviolet detector, the detection wavelength is 280nm, and the first chromatographic peak is the viral protein chromatographic peak.
(6) Preparing stock solution: adding protective agent to prepare stock solution.
4. Preparation of finished product
Adding protective agent to obtain semi-finished product, packaging, and lyophilizing to obtain the final product.
The volume of the protective agent depends on the content of the antigen in the stock solution, the volume of the freeze-drying protective agent needs to be adjusted, and the protein content is not higher than 80 mu g/dose (excluding the content of human serum albumin); 1mL of each dose.
EXAMPLE 3 preparation of the rabies vaccine of the invention
1. Production cell
The production cells are Vero cells adapted to serum-free culture.
2. Poison seed
The production virus seed is rabies virus aG strain.
3. Stock preparation
(1) Cell culture: culturing 1 or more recovered cells in a recovery working cell bank (P140) at 37 +/-1 ℃, amplifying and passaging to (P146) according to the ratio of 1: 3-1: 6, inoculating to a sheet-shaped carrier bioreactor (P147), wherein the inoculation density of the cells in the bioreactor is 3-5 multiplied by 105cells/ml. The formula of the culture medium for culturing the cells is as follows:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(2) Virus inoculation: when the cell density in the bioreactor reaches 1-1.5 × 107cell/ml, inoculating rabies virus (MOl: 1/400-1/100), culturing for 6-8 h after virus infection, and beginning perfusion of the cell maintenance solution.
The maintenance liquid comprises the following components:
the culture method comprises the following steps: perfusion culture is carried out at 33-35 ℃, pH7.50 +/-0.1, dissolved oxygen of 60 +/-20% and rotation speed of 90-110 rpm.
(3) And (3) toxin collection: perfusing and culturing until 2-3 days after the cells are infected with the virus, beginning perfusing to harvest virus liquid, wherein the droplet size of the harvested virus liquid is not less than 6.5LgLD50And (4) the concentration is/ml. Coarse-filtering with a 0.65 μm filter element, and concentrating by 20-30 times with a 300-500 kD membrane module.
(4) Inactivation: adding beta-propiolactone into the concentrated virus harvest liquid according to the volume ratio of 1: 4000, and inactivating the rabies virus at 8 ℃ for 24 +/-2 hours; standing at 37 +/-1 ℃ for 2-3 hours to hydrolyze the beta-propiolactone.
(5) And (3) purification: selecting agarose 4FF as a filler to carry out molecular sieve chromatography purification, taking a phosphate buffer solution with pH of 7.4-7.8 as a mobile phase, wherein the loading amount is 3-8% of the column volume; the detector is an ultraviolet detector, the detection wavelength is 280nm, and the first chromatographic peak is the viral protein chromatographic peak.
(6) Preparing stock solution: adding protective agent to prepare stock solution.
4. Preparation of finished product
Adding protective agent to obtain semi-finished product, packaging, and lyophilizing to obtain the final product.
The volume of the protective agent depends on the content of the antigen in the stock solution, the volume of the freeze-drying protective agent needs to be adjusted, and the protein content is not higher than 80 mu g/dose (excluding the content of human serum albumin); 1mL of each dose.
According to the method for preparing the rabies vaccine, serum or human serum albumin is not added into the maintenance liquid and the cell culture medium, so that the pollution risk of components from animals is reduced, the sensitizers generated by the reaction of the human serum albumin and the beta-propiolactone are avoided, and the safety of the vaccine is integrally improved.
The advantageous effects of the present invention are further illustrated in the form of experimental examples.
Experimental example 1 production efficiency of the Process of the present invention
Rabies vaccine was prepared by the procedure of example 1, and the number of preparations produced per pot was counted, and the titer of each dose was determined and compared with that of the manufacturer A, B, C, D.
The data associated with factory A, B, C, D was verified by asking the factory A, B, C, D and by consulting the china food and drug certification institute official website for the quantity of product on the market per batch.
The results are shown in Table 1. The process of example 1 produced far more preparations than manufacturer A, C, D, with manufacturer B having an incubator volume 8 times that of example 1, but less than 2 times that of example 1. The potency of the preparation obtained in example 1 is also much higher than that of the manufacturer A, B, C.
TABLE 1 comparison of production efficiencies
Note: ND, not detected; the manufacturer A is 0.5 ml/dose, and the rest is 1 ml/dose;
it can be seen that the process of example 1 produces vaccine preparations in large quantities, high titers per preparation, and overall production efficiencies well above the level common in the art.
Experimental example 2 quality control of finished product
The rabies vaccine of example 1 and manufacturer A, B, C was subjected to titer measurement, heat stability test, bovine serum albumin residue, Vero cell DNA residue, and Vero cell protein residue detection. The method comprises the following steps:
1. potency assay
Rabies vaccine titer determination (NIH method) for human is carried out according to the general rules 3503 of the 2015 edition of pharmacopoeia of the people's republic of China:
(1) dilution reference vaccine and test article
The reference vaccine and the test article are respectively diluted to the dilution degree of 1: 25, 1: 125, 1: 625 and the like by sterile PBS (the lyophilized product is re-dissolved and then diluted).
(2) Priming mice
The test article and the reference vaccine with different dilutions are used for immunizing 16 mice with 12-14 g respectively, and 0.5ml is injected into the abdominal cavity of each mouse.
(3) Two-free mouse
Mice were immunized again 7 days after priming using the same protocol.
(4) CVS challenge mice
(5) Calculation of attack virus
Assuming that 31LD is contained per 0.03ml50The viral load of (a) is subjected to intracerebral challenge using a CVS with a viral titer of C (IgLD)500.03ml), Ig31 ≈ 1.50, then the CVS is diluted 10C-1.50Doubling is that every 0.03ml contains 31LD50Viral amount of viral suspension.
(6) Injection mouse
On day 14 after priming, the pre-titrated CVS was diluted to 5-100 LD per 0.03ml with virus diluent50A suspension of virus amounts as challenge virus. Attack in the brain with the attack virus every time0.03ml of mouse; simultaneously diluting the challenge virus to 100、10-1、10-2And 10-3Performing virulence titration, wherein each dilution is not less than 8 mice, placing the cage filled with the mice on a cage frame after injection, observing for 14 days day by day, recording death conditions, and counting the number of the mice which die and show typical rabies brain symptoms after 5 days.
(7) Titer P formula:
dT is the dosage of 1 patient to be tested, ml; dSIs 1 human dose, ml, of the reference vaccine; d is the titer of the reference vaccine, IU/ml.
2. Heat stability test
After standing at 37 ℃ for 28 days, the titer was measured.
Detection of Vero cell protein residue
The residual amount of Vero Cell Proteins was determined by the method (pharmacopoeia of the people's republic of China, 2020 edition, enzyme-linked immunosorbent assay) using Cygnus kit "Vero Cell hosts Cell Proteins".
Vero cell DNA residue detection
4.1 pharmacopoeia method
The culture cell supernatant was not less than 1ml and was subjected to the determination of the residual amount of exogenous DNA (rule 3407) in the pharmacopoeia of the people's republic of China (2015 edition), DNA probe hybridization.
4.2 qPCR method
The test is carried out in 2 steps, wherein the first step is to extract host cell DNA, and the second step is to carry out fluorescence quantitative PCR on the DNA. Taking 100 mul of culture cell supernatant, adding lysis solution to crack and release host cell DNA; adsorbing the released DNA by using magnetic beads, keeping the magnetic beads, and removing a lysate; washing the magnetic beads; and eluting the host cell DNA adsorbed on the magnetic beads by using an eluent, transferring the eluent, and performing fluorescent quantitative PCR. Negative control, blank extraction control, extraction recovery control and standard curve are arranged in the whole test to ensure the accuracy and reliability of the result.
The results are shown in Table 2.
In the aspect of titer, the initial titer of the rabies vaccine prepared by the process of the embodiment 1 is obviously higher than that of other manufacturers, and after a heat stability test, the titer is more preserved and the immunogenicity is stronger; in the aspect of residue, the vaccine of the embodiment 1 has no bovine serum albumin residue, and Vero cell DNA and protein residue are lower than those of other manufacturers, so that the safety is higher.
TABLE 2 comparison of product quality
The results of this experimental example show that the vaccine of example 1 has higher immunogenicity and higher safety, and the overall quality is much higher than the common level in the art.
In conclusion, the method of the invention avoids adding human serum albumin and serum into the cell culture medium and the cell maintenance liquid, increases the safety of the vaccine product, improves the production efficiency and the product quality of the vaccine, and has great application value.
Claims (5)
1. A method for preparing a rabies vaccine for human use is characterized by comprising the following steps:
(1) cell culture: culturing Vero cells to 1-1.5 x 10 in a sheet-shaped carrier bioreactor7After cells/ml, perfusion culture is carried out by using a serum-free culture solution;
(2) virus inoculation: inoculating rabies virus rPV-2061 into cells according to 1/400-1/100 MOI, and after inoculating for 6-8 hours, performing perfusion culture by using cell maintenance liquid;
(3) and (3) toxin collection: harvesting the virus from 2-3 days after virus inoculation to obtain a virus harvesting solution;
(4) inactivation: inactivating the virus with beta-propiolactone;
(5) and (3) purification: purifying the virus by molecular sieve chromatography;
(6) preparing stock solution: adding a protective agent to prepare a stock solution;
the serum-free culture solution comprises the following components:
VP-SFM 17.6g/L，
GlutaxMAX 4mmol/L，
the fructose content is 2.0g/L,
Tricine 2.0g/L；
the cell maintenance liquid is prepared by adding sodium bicarbonate with the final concentration of 2.0g/L on the basis of the serum-free culture liquid.
2. The method of claim 1, wherein: the temperature of perfusion culture in the steps (1) and (2) is 33-35 ℃, the pH is 7.50 +/-0.1, the dissolved oxygen is 60 +/-20%, and the rotating speed is 90-110 rpm.
3. The method of claim 1, wherein: the step (3) and the step (4) comprise a step of concentrating the virus harvest liquid by using an ultrafiltration membrane package;
and/or, the filler purified by the molecular sieve chromatography in the step (5) is agarose gel 4 FF.
4. The method of claim 3, wherein: the concentration multiple of the concentrated virus harvesting solution is 20-30 times.
5. The method of any of claims 1 to 4, wherein: and (6) a freeze-drying step is also included after the step (6), namely, a protective agent is added to prepare a semi-finished product, the protein content of the semi-finished product is ensured not to be higher than 80 mu g/ml, and then freeze-drying is carried out.
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