CN113881644A - Method for removing host DNA in rabies vaccine - Google Patents
Method for removing host DNA in rabies vaccine Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 46
- 229960003127 rabies vaccine Drugs 0.000 title claims abstract description 36
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- 238000003306 harvesting Methods 0.000 claims abstract description 41
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- 229960005486 vaccine Drugs 0.000 claims abstract description 21
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 17
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- 238000002360 preparation method Methods 0.000 claims description 16
- 238000000265 homogenisation Methods 0.000 claims description 15
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- 238000006243 chemical reaction Methods 0.000 claims description 14
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- 238000012258 culturing Methods 0.000 claims description 11
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- 238000006460 hydrolysis reaction Methods 0.000 claims description 10
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 10
- 102100030310 5,6-dihydroxyindole-2-carboxylic acid oxidase Human genes 0.000 claims description 9
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 9
- 101000773083 Homo sapiens 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 claims description 9
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- NKAAEMMYHLFEFN-ZVGUSBNCSA-M sodium;(2r,3r)-2,3,4-trihydroxy-4-oxobutanoate Chemical compound [Na+].OC(=O)[C@H](O)[C@@H](O)C([O-])=O NKAAEMMYHLFEFN-ZVGUSBNCSA-M 0.000 claims description 8
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 7
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- 102000008100 Human Serum Albumin Human genes 0.000 description 16
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- UUEWCQRISZBELL-UHFFFAOYSA-N 3-trimethoxysilylpropane-1-thiol Chemical compound CO[Si](OC)(OC)CCCS UUEWCQRISZBELL-UHFFFAOYSA-N 0.000 description 13
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- 238000001291 vacuum drying Methods 0.000 description 10
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
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- 238000001514 detection method Methods 0.000 description 5
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- 238000012360 testing method Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 3
- 241000711798 Rabies lyssavirus Species 0.000 description 2
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- 238000003672 processing method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229950008679 protamine sulfate Drugs 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20151—Methods of production or purification of viral material
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Abstract
The invention discloses a method for removing host DNA in rabies vaccine, which comprises the steps of firstly selecting Vero cells with clear cell boundaries and few aging cells in the production process, carrying out subculture after cell counting and adjusting to the required cell density, then carrying out virus inoculation, and harvesting virus liquid about 6 days after virus inoculation; on this basis, this application carries out ultrasonic dispersion after mixing virus liquid and EDTA evenly, and EDTA is as metal chelator, can chelate divalent cation in order to disperse DNA, carries out ultrasonic dispersion and high pressure homogeneity processing on this step basis afterwards, and the two combines each other, can cut host DNA for the host DNA chain fracture is broken, with the efficiency of getting rid of that improves follow-up remaining host DNA. According to the scheme, the magnetic silica is introduced, the separation of the magnetic silica and the vaccine can be realized through an external magnetic field, the residual DNA is adsorbed in the process for purification, new impurities do not need to be introduced, the quality of the vaccine is greatly improved, and the practicability is higher.
Description
Technical Field
The invention relates to the technical field of vaccine purification, in particular to a method for removing host DNA in rabies vaccine.
Background
As is known to all, in the process of processing and producing rabies vaccines, cell culture supernatant infected by virus not only contains rabies virus, but also contains host cell fragments and host DNA, and in order to ensure the safety of rabies vaccines, the residual amount of the host DNA of the rabies vaccine (Vero) cells for freeze-dried human use specified by Chinese pharmacopoeia is not higher than 3 ng/dose.
In the prior art, various methods for removing residual DNA of rabies vaccines are developed, such as a method for removing DNA by adding protamine sulfate into a virus harvest solution, and the scheme has serious virus loss and low recovery rate; the actual impurity removal effect of the conventionally adopted gel filtration chromatography cannot meet the regulation of Chinese pharmacopoeia, and the yield of the vaccine is influenced.
Aiming at the technical problem, a method for removing host DNA in rabies vaccine is disclosed to remove residual host DNA without introducing new impurities, so that the quality of the vaccine is greatly improved.
Disclosure of Invention
The present invention is directed to a method for removing host DNA in a rabies vaccine, which solves the problems mentioned above in the background art.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) taking Vero cells, counting, carrying out subculture, discarding cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, carrying out transfer culture, and harvesting virus liquid; mixing virus solution and EDTA (ethylene diamine tetraacetic acid) solution uniformly, performing ultrasonic dispersion, and performing high-pressure homogenization treatment to obtain homogenized harvest solution;
(2) taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
and (3) mixing and stirring the PBS buffer solution and the silicon compound, adjusting the pH, adding the inactivated concentrated solution, centrifuging, collecting the supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
The optimized scheme comprises the following steps:
(1) taking Vero cells, counting, subculturing, discarding cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33-35 ℃ for culturing, and harvesting virus liquid after culturing for 5-7 days; taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 5-10 min, and performing high-pressure homogenization treatment under the conditions that the pressure is 500-1100 bar and the flow rate is 6-10L/h to obtain a homogenized harvest liquid;
(2) taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
and (3) mixing and stirring PBS and a silicon compound for 20-30 min, adjusting the pH value to 7.2, adding the inactivated concentrated solution, centrifuging to collect supernatant, clarifying and filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
In the optimized scheme, in the step (1), the technological parameters of the high-pressure homogenization treatment are as follows: the pressure was 800bar and the flow rate was 8L/h.
In an optimized scheme, in the step (2), the silicon compound is any one of glass fiber, silica gel, nano-silica and magnetic nano-silica.
In an optimized scheme, when the silicon compound is magnetic nano silicon dioxide, the silicon compound is in an external magnetic field during the operation of the step (2).
In an optimized scheme, the strength of the external magnetic field is 4-6 mT.
In an optimized scheme, the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 10-20 min, adding CTAB (cetyl trimethyl ammonium bromide), diethyl ether and ammonia water, mixing and stirring for 20-30 min, adding TEOS (tetraethyl orthosilicate) and MPS (3-mercaptopropyl trimethoxy silane), reacting for 4-5 h at 25-30 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A;
taking the material A, removing a surfactant CATB, drying in vacuum, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 3-4 h at 25-30 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP (4-dimethylaminopyridine), carrying out constant-temperature reaction for 3-4 h in a water bath at 65-70 ℃, washing with deionized water, and drying in vacuum to obtain the magnetic nano-silica.
According to an optimized scheme, the specific steps for removing the surfactant CATB are as follows: and transferring the material A into a mixed solution of ammonium nitrate and ethanol, carrying out constant-temperature treatment at 60-65 ℃ for 20-30 min, carrying out centrifugal washing, and repeating the steps for 2-3 times.
According to an optimized scheme, in the step (1), the power is 200-400W during ultrasonic dispersion.
Compared with the prior art, the invention has the following beneficial effects:
as is well known, in the process of processing and producing rabies vaccines, cell culture supernatant infected by virus not only contains rabies virus, but also contains host cell fragments and host DNA, and in order to ensure the safety of rabies vaccines, the Chinese pharmacopoeia stipulates that the host DNA residue of the rabies vaccine (Vero) cells for freeze-dried human use is not higher than 3 ng/dose; in order to realize safe quality control of the rabies vaccine and remove the host DNA to the greatest extent, the application discloses a method for removing the host DNA in the rabies vaccine so as to optimize the whole production and processing method.
During production, Vero cells with clear cell boundary and less aged cells are selected, after cell counting, subcultured and adjusted to required cell density, and the total number of the cells is controlled to be 3.1 multiplied by 108Then, virus inoculation is carried out, and virus liquid is harvested about 6 days after virus inoculation; in the prior art, virus liquid harvesting is generally carried out for 2-3 times, the culture time can be as long as 12 days, the limited culture parameter of the step is one-time harvesting, and virus liquid inoculated with viruses and cultured for 6 days is harvested, the reason is that the state of cells at the initial stage of maintenance liquid culture is good, the cell metabolic product quantity is small, the pH is relatively stable, the DNA and cell debris quantity released by Vero cells in the culture process is small, and along with the extension of the culture time, although the maintenance liquid can be replaced in the actual production process, the cell aging phenomenon is obvious in the process, and the cell debris and host DNA quantity are obviously increased; the virus fluid is thus harvested according to the procedure parameters disclosed in this application,the method can reduce the subsequent purification difficulty while harvesting the high-titer virus liquid, reduce the host DNA and cell debris contained in the virus liquid, and has higher practicability.
On this basis, this application carries out ultrasonic dispersion after mixing virus liquid and EDTA evenly, and EDTA is as metal chelator, can chelate divalent cation in order to disperse DNA, carries out ultrasonic dispersion and high pressure homogeneity processing on this step basis afterwards, and the two combines each other, can cut host DNA for the host DNA chain fracture is broken, with the efficiency of getting rid of that improves follow-up remaining host DNA.
In the process, ultrasonic power and high-pressure homogenization treatment process parameters are limited, the phenomenon that the subsequent purification removal is influenced due to the fact that DNA fragments are too small and too fine is avoided, the phenomenon that DNA chains are not broken thoroughly is also avoided, and the removal effect cannot be improved; however, in practical operation, the molecular weight of host DNA is different, the sizes of the DNA fragments after being broken in the breaking process are not consistent, and in the subsequent purification process, pure nano-silica is adopted for adsorption according to a conventional method, so that the purification efficiency is low; therefore, the silicon compound is optimized and modified by the application, and the specific preparation process is as follows:
aiming at the problem that the sizes of fragments of the host DNA after being fractured in the previous steps are uneven, components such as CTAB, diethyl ether, ammonia water, TEOS and MPS are adopted, reaction parameters are controlled to realize the outwards-opened mesoporous silica with a radial structure, the mesoporous silica has a high specific surface area, the structure can enable the DNA fragments with different sizes to pass through and be adsorbed, and the host DNA removing effect is more excellent. Meanwhile, magnetic ferroferric oxide is introduced into mesoporous silica, on one hand, the subsequent separation of viruses and silicon compounds is facilitated due to the introduction of magnetism, the introduction of impurities is avoided to the greatest extent, on the other hand, when the silicon compounds adsorb host DNA, the whole operation is carried out in a magnetic field with the magnetic field intensity of 4-6 mT, the magnetic field direction changes along with time, the frequency of the change of the magnetic field direction is 0.5Hz, the magnetic field replaces the conventional ultrasonic dispersion or stirring effect, the adsorption contact between the silicon compounds and the host DNA can be further improved while the influence on the viruses is avoided, and the adsorption effect is improved;
on the basis, sodium hydrogen tartrate is selected to coat the magnetic silicon dioxide, active groups are introduced while the dispersity of the magnetic silicon dioxide is improved, surface amination modification is carried out through ethylenediamine, and host DNA with negative charges is adsorbed to achieve removal of the host DNA.
The invention discloses a method for removing host DNA in rabies vaccine, which has reasonable process design and simple operation, wherein a silicon compound is added in the production process of the vaccine to remove residual host DNA.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 34 ℃ for culture, and harvesting virus liquid after 7 days of culture;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 5min, and performing high-pressure homogenization treatment under the conditions of pressure of 500bar and flow rate of 6L/h to obtain a homogenized harvest liquid; the power is 200W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at a rotating speed of 300r/min, stirring for 20min, adjusting pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, sterilizing, packaging, and lyophilizing to obtain the final product vaccine
In this embodiment, the silicon compound is nano silicon dioxide. The particle size of the silicon compound is 100 nm.
Example 2:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 8min, and performing high-pressure homogenization treatment under the conditions of pressure of 800bar and flow rate of 8L/h to obtain a homogenized harvest liquid; the power is 300W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
In this embodiment, the silicon compound is nano silicon dioxide. The silicon compound has a particle size of 100nm
Example 3:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 35 ℃ for culture, and harvesting virus liquid after culturing for 5 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 10min, and performing high-pressure homogenization treatment under the conditions that the pressure is 1100bar and the flow rate is 10L/h to obtain a homogenized harvest liquid; the power during ultrasonic dispersion is 400W.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 30min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
In this embodiment, the silicon compound is nano silicon dioxide. The silicon compound has a particle size of 100nm
Example 4:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 8min, and performing high-pressure homogenization treatment under the conditions of pressure of 800bar and flow rate of 8L/h to obtain a homogenized harvest liquid; the power is 300W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
The step (2) is in an external magnetic field during operation, and the intensity of the external magnetic field is 4 mT; the direction of the magnetic field changes with time, and the frequency of the change of the direction of the magnetic field is 0.5 Hz.
In this embodiment, the silicon compound is magnetic silicon dioxide. The particle size of the silicon compound is 100 nm; the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 10min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 20min, adding TEOS and MPS, reacting for 5h at 25 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A; wherein the MPS/TEOS volume ratio is 0.08.
Taking a material A, removing a surfactant CATB, transferring the material A into a mixed solution of ammonium nitrate and ethanol during operation, carrying out constant temperature treatment for 30min at 60 ℃, carrying out centrifugal washing, repeating the step for 3 times, carrying out vacuum drying, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 4h at 25 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP, carrying out constant temperature reaction for 4h in a water bath at 65 ℃, washing with deionized water, and carrying out vacuum drying to obtain the magnetic nano-silica.
Example 5:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 8min, and performing high-pressure homogenization treatment under the conditions of pressure of 800bar and flow rate of 8L/h to obtain a homogenized harvest liquid; the power is 300W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
The step (2) is in an external magnetic field during operation, and the intensity of the external magnetic field is 5 mT; the direction of the magnetic field changes with time, and the frequency of the change of the direction of the magnetic field is 0.5 Hz.
In this embodiment, the silicon compound is magnetic silicon dioxide. The particle size of the silicon compound is 100 nm; the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 15min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 25min, adding TEOS and MPS, reacting for 4.5h at 28 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A; wherein the MPS/TEOS volume ratio is 0.08.
Taking a material A, removing a surfactant CATB, transferring the material A into a mixed solution of ammonium nitrate and ethanol during operation, carrying out constant temperature treatment for 25min at 63 ℃, carrying out centrifugal washing, repeating the step for 3 times, carrying out vacuum drying, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 3.5h at 28 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP, carrying out constant temperature reaction for 3.5h in a water bath at 68 ℃, washing with deionized water, and carrying out vacuum drying to obtain the magnetic nano-silica.
Example 6:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 8min, and performing high-pressure homogenization treatment under the conditions of pressure of 800bar and flow rate of 8L/h to obtain a homogenized harvest liquid; the power is 300W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
The step (2) is in an external magnetic field during operation, and the intensity of the external magnetic field is 6 mT; the direction of the magnetic field changes with time, and the frequency of the change of the direction of the magnetic field is 0.5 Hz.
In this embodiment, the silicon compound is magnetic silicon dioxide. The particle size of the silicon compound is 100 nm; the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, performing ultrasonic dispersion for 20min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 30min, adding TEOS and MPS, reacting for 4h at 30 ℃, performing centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A; wherein the MPS/TEOS volume ratio is 0.08.
Taking a material A, removing a surfactant CATB, transferring the material A into a mixed solution of ammonium nitrate and ethanol during operation, carrying out constant temperature treatment for 20min at 65 ℃, carrying out centrifugal washing, repeating the step for 3 times, carrying out vacuum drying, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 3h at 30 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP, carrying out constant temperature reaction for 3h in 70 ℃ water bath, washing with deionized water, and carrying out vacuum drying to obtain the magnetic nano-silica.
Comparative example 1:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
Taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 8min, and performing high-pressure homogenization treatment under the conditions of pressure of 800bar and flow rate of 8L/h to obtain a homogenized harvest liquid; the power is 300W when ultrasonic dispersion is carried out.
(2) Taking the homogenized harvest liquid, concentrating by using an ultrafiltration membrane, and inactivating viruses to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
In this embodiment, the silicon compound is magnetic silicon dioxide. The particle size of the silicon compound is 100 nm; the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 15min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 25min, adding TEOS and MPS, reacting for 4.5h at 28 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A; wherein the MPS/TEOS volume ratio is 0.08.
Taking a material A, removing a surfactant CATB, transferring the material A into a mixed solution of ammonium nitrate and ethanol during operation, carrying out constant temperature treatment for 25min at 63 ℃, carrying out centrifugal washing, repeating the step for 3 times, carrying out vacuum drying, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 3.5h at 28 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP, carrying out constant temperature reaction for 3.5h in a water bath at 68 ℃, washing with deionized water, and carrying out vacuum drying to obtain the magnetic nano-silica.
Comparative example 1 is a control test of example 5, in comparative example 1 no applied magnetic field is introduced, and the remaining process parameters are identical to those of example 5.
Comparative example 2:
a method for removing host DNA in a rabies vaccine, comprising the steps of:
(1) selecting Vero cells with clear cell boundary and few aging cells, subculturing after cell counting, adjusting to required cell density, and controlling total cell number to be 3.1 × 108Abandoning cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33 ℃ for culture, and harvesting virus liquid after culturing for 6 days;
wherein the maintenance liquid is 199 culture solution containing 0.2% human serum albumin, and the preparation method comprises the following steps: 89mL of water for injection and 10mL of 199 mother liquor are taken, stirred and dissolved, 1mL of 20% human serum albumin is added, the pH value is adjusted to 7.8 by sodium bicarbonate, and the mixture is sterilized and filtered by a 0.2-micron filter membrane. Storing at 2-8 deg.C in dark.
And (3) taking the virus liquid, and carrying out high-pressure homogenization treatment under the conditions that the pressure is 800bar and the flow rate is 8L/h to obtain a homogenized harvest liquid.
(2) Taking the homogenized harvest liquid, concentrating by using an ultrafiltration membrane, and inactivating viruses to obtain an inactivated concentrated solution;
mixing 500mL of LPBS and 50g of silicon compound at the rotating speed of 300r/min, stirring for 25min, adjusting the pH to 7.2, adding 10mL of inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
The step (2) is in an external magnetic field during operation, and the intensity of the external magnetic field is 5 mT; the direction of the magnetic field changes with time, and the frequency of the change of the direction of the magnetic field is 0.5 Hz.
In this embodiment, the silicon compound is magnetic silicon dioxide. The particle size of the silicon compound is 100 nm; the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 15min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 25min, adding TEOS and MPS, reacting for 4.5h at 28 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A; wherein the MPS/TEOS volume ratio is 0.08.
Taking a material A, removing a surfactant CATB, transferring the material A into a mixed solution of ammonium nitrate and ethanol during operation, carrying out constant temperature treatment for 25min at 63 ℃, carrying out centrifugal washing, repeating the step for 3 times, carrying out vacuum drying, transferring into sodium hydrogen tartrate, carrying out ultrasonic reaction for 3.5h at 28 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, transferring into ethylenediamine after drying, adding DMAP, carrying out constant temperature reaction for 3.5h in a water bath at 68 ℃, washing with deionized water, and carrying out vacuum drying to obtain the magnetic nano-silica.
Comparative example 2 is a control of example 5, comparative example 2 does not incorporate EDTA solution and the remaining process parameters are consistent with example 5.
In the above examples of the present application, the amount of the nano-silica or magnetic silica is 50g, the PBS buffer solution is 500mL, and the inactivated concentrated solution of the virus is 10mL, and the final concentration is 0.026 g/L.
And (3) detection test:
1. the DNA residue of Vero cells in rabies vaccine is detected according to the PCR method specified in the three annexes of the 2020 edition of the pharmacopoeia of the people's republic of China: the operation is carried out according to the instructions of the advanced kit and the detection kit, and the DNA content of each test group is determined.
The detection data are as follows:
| item | Example 1 | Example 2 | Example 3 | Example 4 | Example 5 |
| Host DNA (ng/ml) | 1.1 | 0.9 | 1.0 | 0.5 | 0.2 |
| Removal rate | 86.3％ | 88.8％ | 87.5％ | 93.8％ | 97.5％ |
| Item | Example 6 | Comparative example 1 | Comparative example 2 | ||
| Host DNA (ng/ml) | 0.4 | 0.7 | 0.4 | ||
| Removal rate | 95％ | 91.3％ | 95％ |
2. The finished vaccines prepared in the examples 1-6 and the comparative examples 1-2 are detected, the detection standard is appendix three parts of pharmacopoeia of the people's republic of China 2020 edition, wherein the sterility test, the mycoplasma test and the abnormal toxicity test all accord with the third part standard of the pharmacopoeia of the 2020 edition, the detection result is qualified, and the prepared vaccine accords with the safety standard.
And (4) conclusion: the invention discloses a method for removing host DNA in rabies vaccine, which has reasonable process design and simple operation, wherein a silicon compound is added in the production process of the vaccine to remove residual host DNA.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for removing host DNA in a rabies vaccine, comprising: the method comprises the following steps:
(1) taking Vero cells, counting, carrying out subculture, discarding cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, carrying out transfer culture, and harvesting virus liquid; uniformly mixing virus solution and EDTA solution, performing ultrasonic dispersion, and performing high-pressure homogenization treatment to obtain homogenized harvest solution;
(2) taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
mixing PBS and silicon compound, stirring, adjusting pH, adding the inactivated concentrated solution, centrifuging, collecting supernatant, clarifying, filtering, sterilizing, packaging, and lyophilizing to obtain the final product vaccine.
2. The method for removing host DNA in a rabies vaccine according to claim 1, wherein: the method comprises the following steps:
(1) taking Vero cells, counting, subculturing, discarding cell growth liquid, inoculating virus seeds, supplementing maintenance liquid, transferring to 33-35 ℃ for culturing, and harvesting virus liquid after culturing for 5-7 days; taking virus liquid and EDTA solution, mixing uniformly, performing ultrasonic dispersion for 5-10 min, and performing high-pressure homogenization treatment under the conditions that the pressure is 500-1100 bar and the flow rate is 6-10L/h to obtain a homogenized harvest liquid;
(2) taking the homogenized harvest solution, concentrating by using an ultrafiltration membrane, and then performing virus inactivation and hydrolysis to obtain an inactivated concentrated solution;
and (3) mixing and stirring PBS and a silicon compound for 20-30 min, adjusting the pH value to 7.2, adding the inactivated concentrated solution, centrifuging to collect supernatant, clarifying and filtering, preparing for sterilization, subpackaging and freeze-drying to obtain the finished vaccine.
3. The method for removing host DNA in a rabies vaccine according to claim 2, wherein: in the step (1), the technological parameters of the high-pressure homogenization treatment are as follows: the pressure was 800bar and the flow rate was 8L/h.
4. The method for removing host DNA in a rabies vaccine according to claim 2, wherein: in the step (2), the silicon compound is any one of glass fiber, silica gel, nano silicon dioxide and magnetic nano silicon dioxide.
5. The method for removing host DNA in rabies vaccine according to claim 4, wherein: when the silicon compound is magnetic nano silicon dioxide, the silicon compound is in an external magnetic field during the operation of the step (2).
6. The method for removing host DNA in rabies vaccine according to claim 5, wherein: the strength of the external magnetic field is 4-6 mT.
7. The method for removing host DNA in rabies vaccine according to claim 5, wherein: the preparation steps of the magnetic nano silicon dioxide are as follows:
taking nano ferroferric oxide, ethanol and deionized water, carrying out ultrasonic dispersion for 10-20 min, adding CTAB, diethyl ether and ammonia water, mixing and stirring for 20-30 min, adding TEOS and MPS, reacting for 4-5 h at 25-30 ℃, carrying out centrifugal filtration, washing with ethanol and deionized water in sequence, and drying to obtain a material A;
and taking the material A, removing a surfactant CATB, drying in vacuum, transferring to sodium hydrogen tartrate, carrying out ultrasonic reaction for 3-4 h at 25-30 ℃, carrying out magnetic separation for solid-liquid separation, washing with deionized water, drying, transferring to ethylenediamine, adding DMAP, reacting for 3-4 h at a constant temperature in a water bath at 65-70 ℃, washing with deionized water, and drying in vacuum to obtain the magnetic nano-silica.
8. The method for removing host DNA in rabies vaccine according to claim 7, wherein: the specific steps for removing the surfactant CATB are as follows: and transferring the material A into a mixed solution of ammonium nitrate and ethanol, carrying out constant-temperature treatment at 60-65 ℃ for 20-30 min, carrying out centrifugal washing, and repeating the steps for 2-3 times.
9. The method for removing host DNA in a rabies vaccine according to claim 2, wherein: in the step (1), the power is 200-400W during ultrasonic dispersion.
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