CN111249456A - Purification method of rabies virus inactivated vaccine - Google Patents
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Abstract
The invention relates to a purification method of a rabies virus inactivated vaccine, which comprises the following steps: (1) inoculating and culturing a rabies vaccine strain by taking the Vero cell as a virus host, and harvesting a virus concentrated solution; (2) inactivating viruses; (3) preparing purified inactivated rabies vaccine by combining molecular sieve chromatography with anion exchange chromatography; the method does not add any chemical purifying agent into the vaccine, has simple operation and good process continuity, and can avoid the dilution of the virus antigen purification process.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a purification method of a rabies virus inactivated vaccine.
Background
Rabies (Rabies) is a natural epidemic-origin or animal-origin human and animal co-suffering acute infectious disease caused by Rabies virus, has wide popularity and extremely high lethality rate, and poses serious threat to the life and health of people. Vaccination is currently still the most effective method for the prevention and control of rabies. Since the first lewis pasteur invention in 1885 used rabies vaccine, rabies vaccine developed from early animal nervous tissue vaccine, avian embryo vaccine, crude vaccine in cell culture, to the current purified vaccine cultured by primary hamster kidney cells, primary chicken embryo cells, primary duck embryo cells, african green monkey kidney cells, rhesus fetal diploid cells, human diploid cells.
Before 1980, the adult sheep brain tissue vaccine is produced and used in China, adverse reactions are large after inoculation, particularly, allergic encephalomyelitis caused by brain tissues is particularly serious, and meanwhile, the immune effect is not satisfactory enough. In order to solve the problems, the research institute of Wuhan biological products of the Ministry of health, the institute of medicine biological products of China, Changchun of the Ministry of health, the institute of Lanzhou biological products and other units start the research and development work of cell culture vaccines in cooperation in 1965 in China, and finally the vaccines are applied in 1980 and replace sheep brain vaccines. The freeze-dried rabies vaccine (Vero cells) for human use occupies most of rabies vaccine markets in China at present due to the comprehensive advantages of the freeze-dried rabies vaccine (Vero cells) in the aspects of production process, economic benefit, quality and the like.
Vero cell as a passage cell, the DNA of the Vero cell acts with the normal cells of human body to cause the normal cells of human body to generate mutation, thereby causing the risk of canceration[1,2]. Although 130-150 th-generation Vero cells are considered to be non-tumorigenic internationally at present and can be applied to vaccine production, the growth promoting proteins and cell matrixes such as cell residual proteins, potential viruses and cell residues cause serious worry about the mass use of subculture cell vaccines, so that related standards for cell residual quantity in biological products are established by a plurality of organization tissues in the world at present in order to ensure the safety of vaccines[3]. The existing pharmacopoeia (third edition in 2015) of China makes clear regulations on the residual amount of host cell substances in a biological product, and the residual amount of Vero cell DNA is not higher than 100 pg/dose (the first method of 3407); the total protein content is not higher than 80 mug/dose (Lowry method); the residual amount of Vero cell host protein (HCP) is not higher than 4 mu g/dose (adopting an enzyme-linked immunosorbent assay); bacterial endotoxin was not higher than 25 EU/dose (gel limit experiment).
In the early stage, rabies vaccine products were microfiltered to remove tissue debris, shed cells or cell debris using microfiltration membrane filtration techniques. Subsequently, in order to improve the titer of the vaccine, an ultrafiltration concentration technology is added, and some small molecular impurities are also removed in the ultrafiltration process. The sucrose zonal ultracentrifugation purification process established in the seventies of the last century and the gel filtration column chromatography purification technology established in the nineties further improve the purity of the product. After this time, there was no major improvement in the purification process of human rabies vaccine preparations. The purification process of various rabies vaccine products at home and abroad is almost the same, and the purification process sequentially comprises microfiltration clarification, ultrafiltration concentration, sucrose density gradient centrifugation or gel filtration column chromatography.
With the continuous improvement of the quality requirement of the vaccine, people can purify the rabies vaccineAttempts to improve the art have never been stopped, and to summarize the previous attempts to purify rabies virus by zonal centrifugation after inactivation with β -propiolactone[4](ii) a Treating the concentrated virus solution by anion exchange chromatography, cation exchange chromatography, and metal chelating affinity chromatography[5](ii) a Protamine sulfate-agarose gel chromatography and molecular sieve chromatography are used for purifying virus liquid successively[6](ii) a Treating the concentrated virus solution with ultrasonic wave with specific wavelength, purifying by molecular sieve chromatography to obtain a first purified solution, and repeating the above steps to obtain a second purified solution[7](ii) a Before virus inoculation, the production cells are washed by PBS solution containing sodium butyrate and protamine sulfate, and virus harvest liquid is purified by molecular sieve composite ion chromatography[8](ii) a Sequentially treating the ultrafiltered concentrated virus solution by cation exchange chromatography, nuclease and sucrose gradient ultracentrifugation to realize purification[9](ii) a Purifying virus liquid by anion exchange chromatography and hydroxyapatite chromatography in sequence which can be interchanged[10]And the like.
The above methods have so far suffered from some drawbacks, to a greater or lesser extent. For example, the ultrasonic treatment inevitably causes certain damage to the activity of the virus target protein and influences the yield of vaccine products; moreover, ultrasonic waves can alter the original structure of the material, potentially complicating the composition of the impurities. The density gradient centrifugation operation is complicated, the workload is large, the equipment is expensive, the large-scale industrial production is not suitable, and the bacterial contamination risk of non-terminal sterilized products is greatly increased; protamine sulfate binds to DNA and part of virus glycoprotein, causing great activity loss[11]. Li Xue et al[12]The effect of purifying rabies vaccine by the novel composite medium Capto Core700 is studied, and the impurity removal capability of the novel composite medium Capto Core700 is not greatly different from that of the traditional Sepharose series gel purification method, and the novel composite medium Capto Core700 mainly has advantages in purifying sample volume. Zeng long bright, etc[13]Attempts to perform a small pilot vaccine manufacturing process using Sepharose CL-6B and DEAE exchange resins as media were made as early as 1997, but the virus yield and scale-up of the process have not been explored further。
Reference documents:
1.Khan A S.Characterization and Qualification of Cell Substrates forManufacturing Viral Vaccines in the United States[J].Bioprocessing Jouranl2009,3(8):8-11.
2.Guidance for Industry Characterization and qualification of CellSubstrates and Othe Biological Maerials Used in the Production of ViralVaccine for Infectious Disease indications,U.S.Department of Health and HumanServices FDA,CBER,February 2010.
3. royal orchid, royal military, about the problem of controlling the quality of the residual DNA of biological products [ J ]. Chinese New medicine journal, 2011,20(8):678-687.
4.Wikto T J,Fanget B J,Fournier P.Process for the large scaleproduction of rabies vaccine.US4664912A.1987.05.12.
5.Fanget B,Francon A.Method for purifying viruses by chromatograpHy.US6008036A.1990.12.18.
6. Zhangiue, Huanglin, Welan bin, et al, established the purification process of rabies vaccine with VERO cells [ J ]. journal of preventive medicine information, 2003,19(03): 198-.
7. Zhang Ying, Wangkai, Suwenquan, etc. the method for removing hetero protein and host DNA from vaccine product is CN102018958A.2011.04.20.
8. Wangxuyun, Zhao Xiao' e, Zhuxiuyuan, etc. A method for preparing rabies vaccine for human use is CN104027800A.2014.09.10.
Methods for purifying rabies viruses, such as v. fabry, c. roca, p. lifal, etc. cn105012947a.2015.11.04.
10. The invention discloses unfamiliar inventors, a production method of rabies vaccine, CN108524929A.2018.09.14.
11. Zhangiue, Huanglin, Welan bin, et al, established the purification process of rabies vaccine using VERO cells [ J ]. journal of preventive medicine information 2003,19(03): 198-.
12. Lexu, Xuwei, Hai et al, applied to the complex purification medium Capto Core700 for the purification of rabies vaccine [ J ]. J.J.China J.Biometrics 2018,31(05):538-542.
13. Zeng Rong Bong, Song's family Li, Gu ming, etc. Vero cell rabies vaccine development [ J ]. Chinese virology 1997,12(1):43-49.
14. National pharmacopoeia committee, chinese pharmacopoeia (three) [ S ]. beijing, chinese medical science and technology publisher, 2015:147.
15. Study on constant brightness, Liu Xiao Zhi, high-health rabies virus glycoprotein and its neutralizing antibody advances [ J ] biotechnological advances, 2013,3(5): 317-.
16.Lafon M,Wiktor T,Macfalan R I.Antigenic sites onthe CVS rabiesvirus glycoprotein:analysis with monoclonalantibodies[J].J Gen Virol,1983,64(4):843-851.。
Disclosure of Invention
The invention aims to realize the aims of not obviously influencing the drug effect of the vaccine product and effectively removing various impurities.
The invention provides a production method for massively purifying and concentrating a virus solution by combining molecular sieve chromatography with anion exchange chromatography so as to obtain a rabies vaccine meeting the quality standard of the current pharmacopoeia.
The invention firstly relates to a production method of inactivated rabies vaccine, which comprises the following steps:
(1) inoculating and culturing a rabies vaccine strain by taking the Vero cell as a virus host, and harvesting a virus concentrated solution;
(2) inactivating viruses;
(3) and (3) preparing the purified inactivated rabies vaccine by combining molecular sieve chromatography with anion exchange chromatography.
The specific steps of the step (1) are as follows:
1) culturing the recovered single-layer Vero cells at 37 ℃ to form uniform single-layer cells, wherein the used culture medium is an M199 cell culture medium containing 5% bovine serum;
2) inoculating the cultured Vero cells into a final reactor, and carrying out cell culture in a bioreactor with the volume of 75L-300L; the culture parameters are temperature 36.5 + -1 deg.C, pH 7.25 + -0.2, and pO230.0-80.0% and perfusion rate of 1-5 reactors per 24 hours; the number of cells after 96-120 hours of culture should reach (4.0-10.0) × 106cells/ml；
3) To the above step 2) Inoculating rabies virus seeds into the Vero cells after medium culture according to the MOI of 0.01-0.1, wherein the culture parameters are the temperature of 33-37 ℃, the pH value of 6.8-8.0 and the pO230.0-80.0% and the perfusion rate is 1-5 reactor working volumes/24 hours;
4) after 3 days of virus culture, starting to harvest virus liquid, continuously collecting for 4-6 days, merging the virus liquid, clarifying by a filter element with the aperture of 0.2-0.45 mu m, and concentrating by a 300KD membrane package by 40-80 times to obtain virus concentrated solution, wherein the total protein of the obtained virus liquid is not more than 10 mg/ml;
the specific steps of the step (2) are as follows:
1) adding β -propiolactone (β -propiolactone stock solution) according to the proportion of 1: 4000, and inactivating for 48-72 hours at the temperature of 2-8 ℃;
2) then hydrolyzing for 2-4 hours at 37 ℃ to ensure that β -propiolactone is completely hydrolyzed;
the specific steps of the step (3) are as follows: first, molecular sieve chromatography is carried out, and then anion exchange chromatography is carried out
The molecular sieve chromatography technical parameters are as follows:
1) chromatographic medium Sepharose 4Fast Flow, the height of the chromatographic column is 70-80 cm;
2) the pH value of the balance and elution buffer solution is 6-60 mM PBS (50-500 mM NaCl) with the pH value of 7.2-8.2, and the preferred balance and elution buffer solution is 12-50 mM PBS (100-400 mM NaCl) with the pH value of 7.4-7.9;
3) the dosage of the equilibrium buffer solution is 7.2-8.2 of the effluent liquid after the column, preferably, the dosage of the equilibrium buffer solution is 1.7-2.3 column volumes;
4) the sample loading volume is 3-8% of the column volume;
5) the sampling and elution flow rate is 30-45 cm/h, and preferably, the sampling and elution flow rate is 35-40 cm/h;
6) detecting an absorption value at the wavelength of 280nm by using an ultraviolet detector, and collecting a first elution peak to obtain a crude pure vaccine;
the anion exchange chromatography technical parameters are as follows:
1) chromatography medium Q SepharoseTMXL, wherein the height of the chromatographic column is 20-40 cm;
2) the balance buffer solution is 6-60 mM PBS (50-500 mM NaCl) with pH 7.2-8.2, the elution buffer solution is 6-60 mM PBS (50-1000 mM NaCl) with pH 6.8-7.8, preferably, the balance buffer solution is 12-50 mM PBS (100-400 mM NaCl) with pH 7.4-7.9, and the elution buffer solution is 12-50 mM PBS (300-800 mM NaCl) with pH 7.1-7.6;
3) the sample loading volume is 15-40% of the column volume;
4) the sampling and elution flow rate is 30-45 cm/h, and preferably 35-40 cm/h;
5) the amount of the balance buffer solution is 8-12 column volumes;
6) and detecting the absorption value at the wavelength of 280nm by using an ultraviolet detector, and collecting a first elution peak to obtain a purified inactivated vaccine product.
The invention has the beneficial effects that:
(1) on the premise of not adding any chemical and biological substances into the vaccine product and ensuring the original structure of the vaccine antigen, the difference between the physical and chemical properties of the target protein and impurities is fully utilized, and the foreign protein and host DNA can be effectively removed;
(2) the operation is relatively simple, the pollution risk is low, and a double-flow-path full-automatic production liquid chromatography system can be combined to realize the series connection of two purification processes, so that the process continuity is improved, the downstream process operation is further simplified, and the working efficiency is improved;
(3) the binding capacity of ion exchange chromatography is strong, so that the virus antigen can be prevented from being diluted, and sufficient operation space is provided for preparing a semi-finished product.
Drawings
FIG. 1, sample pattern after molecular sieve chromatography
FIG. 2 sample profile after ion exchange chromatography
FIG. 3 shows the result of transmission electron microscopy on the viral particles in the sample after molecular sieve chromatography
FIG. 4 shows the result of TEM examination of virus particles in the sample after two-stage chromatography
Detailed Description
Example 1 production of rabies vaccine and chromatographic purification
1. Cell and virus seed
Vero cells: from American Standard culture Collection (ATCC CCL-81), generation 134. The rabies vaccine chamber of the company is used for passage to establish a main cell bank and a working cell bank, and the generation time of the production cells is not more than 150.
Rabies virus strain CTN-1V: purchased from china institute for drug and biological products, 23 rd generation. The rabies vaccine is passaged in a rabies vaccine room of the company, a main seed bank and a working seed bank are established, the batch passage of the working seeds for production does not exceed 35 generations, and the virus titer is 7.5-8.0 LgLD 50/ml.
2. Vero cell culture
(1) Taking 1 or more cell tubes in the same batch in the working cell bank, and recovering and amplifying to obtain 1 batch of virus-inoculated cells.
(2) Mixing the recovered monolayer cells with 0.25% trypsin and 0.02% EDTA (ethylene diamine tetraacetic acid) mixed digestive juice or TrypLETMDigesting, dispersing into uniform cells, adding M199 cell culture solution containing 5% bovine serum, and culturing at 37 deg.C to obtain uniform monolayer cells.
(3) After passage to a certain generation according to the method in the step (2), the cells can be cultured in a bioreactor with the volume of 75L-300L by guiding a tank or directly inoculating the cells into a final reactor; the culture parameters are temperature 36.5 + -1 deg.C, pH 7.25 + -0.2, and pO230.0-80.0% and perfusion rate of 1-5 reactors per 24 hours; culturing for 96-120 hours until the cell number reaches (4.0-10.0) × 106cells/ml。
3. Virus inoculation and culture
Inoculating rabies virus seeds into the Vero cell culture equipment cultured in the step 2 according to the MOI of 0.01-0.1, wherein the culture parameters are the temperature of 33-37 ℃, the pH value of 6.8-8.0 and the pO230.0-80.0% and the perfusion rate is 1-5 reactor working volumes/24 hours.
4. Harvesting of viruses
(1) The virus liquid is harvested after 3 days of virus culture, and the collection amount of the virus liquid is the same as the perfusion amount.
(2) According to the needs and the actual conditions, the virus liquid can be continuously harvested for more than 5 days, and the virus liquid harvested every day is single virus harvesting liquid.
5. Clarifying and concentrating virus liquid
And (3) independently combining multiple virus harvesting solutions produced by the same batch of cells into one batch under the strict aseptic operation condition, clarifying by a filter element with the aperture of 0.2-0.45 mu m, and concentrating by 40-80 times by a 300KD membrane package to obtain a virus concentrated solution.
6. Inactivation of viruses
(1) Adding β -propiolactone solution according to the proportion of 1: 4000, and inactivating for 48-72 hours at the temperature of 2-8 ℃;
(2) then hydrolyzing for 2-4 hours at 37 ℃ to ensure that β -propiolactone is completely hydrolyzed.
7. Purification of virus liquid
7.1 molecular Sieve chromatography
(1) Preparation of chromatography column
Before purification, the column loaded with Sepharose 4FastFlow chromatography medium was washed with a 0.5M NaOH solution 1.5 to 2 times the column volume and sterilized.
(2) Chromatography operations
After the chromatographic column is balanced by 1.7-3 times of column volume of PBS buffer solution, loading a virus concentrated solution, wherein the loading volume is 3-8% of the column volume, the loading and elution linear flow velocity is controlled at 30-45 cm/h, and collecting a first elution peak (the wavelength of an ultraviolet detector is 280nm) to obtain an intermediate product 1 (figure 1).
7.2 ion exchange chromatography
(1) Preparation of chromatography column
Before purification, a Q Sepharose (TM) XL chromatographic column is regenerated by using 0.8-1.5M NaCl solution with 1 column volume, and then is cleaned and sterilized by using 1M NaOH solution with 1.5-2 times of column volume.
(2) Chromatography operations
The chromatographic column is balanced by PBS buffer solution with 8-12 times of column volume, then the intermediate product 1 is loaded, the loading volume is 15-40% of the column volume, the loading and elution linear flow velocity is controlled at 30-45 cm/h, and a first elution peak (the wavelength of an ultraviolet detector is 280nm) is collected to obtain a target product (figure 2).
Example 2 evaluation of the Effect of purified vaccine
1. Detection of vaccine Properties
Am according to ChinaPharmacopeia three parts (2015 edition)[14]Detecting the content of host cell protein by using a Vero cell remnant protein detection kit, and calculating the removal rate of the host cell protein (table 1); detecting the residual amount of the host cell DNA by a DNA probe hybridization method, and determining whether the residual amount of the host cell DNA meets the quality standard; quantitative detection is carried out on the DNA by a qPCR method, and the host cell DNA removal rate is calculated (table 2); detecting the content of the rabies virus glycoprotein by an ELISA method, and calculating the recovery rate of the glycoprotein (table 3); the Lowry method measures the total protein content and calculates the protein removal rate (table 4). The results are as follows:
TABLE 1 host cell protein content assay results
TABLE 2 detection of host cell DNA content
TABLE 3 detection of glycoprotein content
TABLE 4 Total protein assay results
2. Morphological testing of vaccines
As the main protective antigen of rabies vaccine, the content and the spatial conception of 3 main antigen sites of rabies glycoprotein determine the titer of the vaccine[15,16]. In actual production, rabies virus produces free small-molecule glycoprotein particles during the replication and assembly process in cells, and the small-molecule glycoprotein particles can be detected by immune experiments, but are not bound on complete virus particles, so the immunogenicity is low, and the immunity of patients cannot be caused. Thus, free small molecule glycoprotein particles are purifiedThe removal can improve the relative content of the effective components of the vaccine and eliminate the interference of impurities. In the present study, the morphology of the virus particles was observed by a transmission electron microscope, and the conformational integrity of the active ingredients after molecular sieve chromatography and after two-stage chromatography was investigated, and the results are shown in fig. 3 and 4, respectively, which indicates that the content of intact rhabdovirus particles in the purified virus solution after two-stage chromatography is significantly higher than that of the traditional single-step molecular sieve chromatography.
3. Vaccine finished product quality detection
The vaccine preparation prepared by using the purified virus solution as a raw material is subjected to quality detection according to a method of a product detection regulation of 'rabies vaccine (Vero cells) for freeze-dried human' recorded in the three parts of the pharmacopoeia of the people's republic of China' 2015 or a related method specified by the general rules of the pharmacopoeia, and is compared with the similar product standard of the current edition of national pharmacopoeia (Table 5).
TABLE 5 vaccine end product testing
Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
Claims (7)
1. A method for producing inactivated rabies vaccine, comprising the following steps:
(1) inoculating and culturing a rabies vaccine strain by taking the Vero cell as a virus host, and harvesting a virus concentrated solution;
(2) inactivating viruses;
(3) and (3) preparing the purified inactivated rabies vaccine by combining molecular sieve chromatography with anion exchange chromatography.
2. The method according to claim 1, wherein the specific steps of step (1) are as follows:
1) culturing the recovered single-layer Vero cells into uniform single-layer cells by using a cell culture medium containing bovine serum;
2) inoculating the cultured Vero cells into a final reactor, and carrying out bioreactor cell culture;
3) inoculating and culturing rabies virus seeds into the Vero cells cultured in the step 2);
4) and (3) starting to harvest virus liquid after virus inoculation and culture for 3 days, continuously collecting for 4-6 days, combining the virus liquid, concentrating the virus liquid to obtain virus concentrated liquid, wherein the total protein of the harvested virus liquid is not more than 10 mg/ml.
3. The method of claim 2,
the parameters of the bioreactor cell culture are as follows:
the volume of the bioreactor is 75L-300L;
the culture parameters are as follows: temperature 36.5 + -1 deg.C, pH 7.25 + -0.2, pO230.0-80.0%, perfusion rate of 1-5 reactors working volume/24 hours, and cell number after 96-120 hours of culture to reach (4.0-10.0) × 106cells/ml；
The parameters for inoculating the rabies virus seeds are as follows:
inoculating rabies virus seeds according to the MOI of 0.01-0.1;
the culture parameters are as follows: at a temperature of 33 to 37 ℃, a pH of 6.8 to 8.0, pO230.0-80.0% and the perfusion rate is 1-5 reactor working volumes/24 hours;
the method for concentrating the virus liquid comprises the following steps: clarifying by a filter element with the aperture of 0.2-0.45 mu m, and concentrating by 40-80 times by a 300KD membrane package.
4. The method according to claim 1 or 2, wherein the specific steps of step (2) are:
1) adding β -propiolactone stock solution according to the proportion of 1: 4000, and inactivating the mixture for 48 to 72 hours at the temperature of 2 to 8 ℃;
2) then hydrolyzing for 2-4 hours at 37 ℃.
5. The method according to claim 1 or 2, wherein the specific steps of step (3) are: first, molecular sieve chromatography is performed, followed by anion exchange chromatography.
6. The method of claim 5, wherein the molecular sieve chromatographic parameters are:
1) chromatographic medium Sepharose 4Fast Flow, the height of the chromatographic column is 70-80 cm;
2) the pH value of the balance and elution buffer solution is 6-60 mM PBS (50-500 mM NaCl) with the pH value of 7.2-8.2, and the preferred balance and elution buffer solution is 12-50 mM PBS (100-400 mM NaCl) with the pH value of 7.4-7.9;
3) the dosage of the equilibrium buffer solution is 7.2-8.2 of the effluent liquid after the column, preferably, the dosage of the equilibrium buffer solution is 1.7-2.3 column volumes;
4) the sample loading volume is 3-8% of the column volume of the virus inactivation liquid obtained in the step (2);
5) the sampling and elution flow rate is 30-45 cm/h, and preferably, the sampling and elution flow rate is 35-40 cm/h;
6) and detecting the absorption value at the wavelength of 280nm by using an ultraviolet detector, and collecting a first elution peak to obtain the crude pure vaccine.
7. The method of claim 5, wherein the anion exchange chromatography parameters are:
1) chromatography medium Q SepharoseTMXL, wherein the height of the chromatographic column is 20-40 cm;
2) the balance buffer solution is 6-60 mM PBS (50-500 mM NaCl) with pH 7.2-8.2, the elution buffer solution is 6-60 mM PBS (50-1000 mM NaCl) with pH 6.8-7.8, preferably, the balance buffer solution is 12-50 mM MPBS (100-400 mM NaCl) with pH 7.4-7.9, and the elution buffer solution is 12-50 mM PBS (300-800 mM NaCl) with pH 7.1-7.6;
3) the sample loading sample is a crude pure vaccine after chromatography, and the sample loading volume is 15-40% of the column volume;
4) the sampling and elution flow rate is 30-45 cm/h, and preferably 35-40 cm/h;
5) the amount of the balance buffer solution is 8-12 column volumes;
6) and detecting the absorption value at the wavelength of 280nm by using an ultraviolet detector, and collecting a first elution peak to obtain a purified inactivated vaccine product.
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