CN110129287B - Porcine pseudorabies virus dual ultrafiltration system and purification method - Google Patents
Porcine pseudorabies virus dual ultrafiltration system and purification method Download PDFInfo
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- CN110129287B CN110129287B CN201910401463.1A CN201910401463A CN110129287B CN 110129287 B CN110129287 B CN 110129287B CN 201910401463 A CN201910401463 A CN 201910401463A CN 110129287 B CN110129287 B CN 110129287B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
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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
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16751—Methods of production or purification of viral material
Abstract
The invention relates to a porcine pseudorabies virus dual ultrafiltration system and a purification method, belonging to the technical field of virus purification. The porcine pseudorabies virus dual ultrafiltration system comprises an antigen mixing tank, a first concentration tank and a second concentration tank which are connected in sequence; a primary clarifying filter and a secondary microfilter are arranged between the antigen mixing tank and the first concentrating tank; a heavy ultrafilter is arranged between the first concentrating tank and the second concentrating tank; the outlet of the second concentration tank is provided with a double ultrafilter; the porcine pseudorabies virus dual ultrafiltration system further comprises a PBS storage tank and an alkali tank which are communicated with the antigen mixing tank, the first concentration tank and the second concentration tank, and the PBS storage tank and the alkali tank are used for cleaning and sterilizing the system on line. The system can obviously reduce insoluble solids, soluble hetero proteins, endotoxin and the like in the porcine pseudorabies virus liquid, reduce the immune side reaction, improve the purity of virus antigens and the quality of vaccine finished products, and has simple process and low cost.
Description
Technical Field
The invention relates to the technical field of virus purification, and in particular provides a porcine pseudorabies virus dual ultrafiltration system and a purification method.
Background
Pseudorabies (Porcine Pseudorabies) is an acute infectious disease caused by pseudorabies virus (Pseudorabies Virus, PRV) and occurring in various domestic animals and wild animals, and has fever, itching and encephalomyelitis as main pathogenesis symptoms. Adult pigs often show recessive infections and pregnant sows can cause abortion, stillbirth and respiratory disease symptoms after infection. In addition to the neurological symptoms that occur after infection of newborn piglets, the digestive system can also be affected. The disease is distributed worldwide, pigs are primary infectious hosts of the pathogen, are long-term reservoirs and detoxifiers of viruses, play an important role in the transmission of pseudorabies, and the harm caused by the pseudorabies to pig farming is inferior to swine fever. The spread and spread of pseudorabies causes huge losses to the aquaculture industry, and the economic losses caused by pseudorabies annually worldwide are as high as billions of dollars, so the disease attracts great attention from countries around the world.
At present, the porcine pseudorabies virus liquid generally adopts continuous flow centrifugation, dead-end filtration, conventional ultrafiltration, chromatography and other methods. Continuous flow centrifugation and dead-end filtration have better removal effects on insoluble solids (e.g., cell debris, macromolecular protein polymers, etc.), but are general for soluble small molecule hybrid proteins; the chromatographic technique can obtain virus liquid with extremely high purity, but the corresponding cost is higher, and the production efficiency is not high.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a porcine pseudorabies virus dual ultrafiltration system and a purification method.
In order to solve the technical problems, the invention provides the following technical scheme:
in one aspect, the invention provides a porcine pseudorabies virus dual ultrafiltration system, which comprises an antigen mixing tank, a first concentration tank and a second concentration tank which are connected in sequence;
a primary clarifying filter and a secondary microfilter are arranged between the antigen mixing tank and the first concentrating tank;
a heavy ultrafilter is arranged between the first concentrating tank and the second concentrating tank; the outlet of the second concentration tank is provided with a double ultrafilter;
the porcine pseudorabies virus dual ultrafiltration system further comprises a PBS storage tank and an alkali tank which are communicated with the antigen mixing tank, the first concentration tank and the second concentration tank, and the PBS storage tank and the alkali tank are used for cleaning and sterilizing the system on line.
Further, a sterilizing filter element is arranged at the outlet of the PBS storage tank and is communicated with the first concentration tank, the second concentration tank, the primary ultra-filter, the dual ultra-filter and the alkali tank through a primary clarification filter and a secondary micro-filter.
Preferably, the primary clarifying filter is a filter element with a pore diameter of 50 μm;
the secondary micro-filter is a filter element with the aperture of 0.45 mu m;
the one-weight ultrafilter is a tangential flow ultrafiltration membrane bag with the aperture of 0.2 mu m;
the double ultrafilter is a tangential flow ultrafiltration membrane bag with a 300kDa pore size.
Further, compressed air inlets are formed in the tops of the PBS storage tank, the antigen mixing tank, the first concentration tank, the second concentration tank and the alkali tank;
further, the first concentration tank is also communicated with a first peristaltic pump;
the second concentration tank is also communicated with a second peristaltic pump.
On the other hand, the invention also provides a purification method of the porcine pseudorabies virus double ultrafiltration, and the porcine pseudorabies virus double ultrafiltration system is adopted.
Further, the purification method of the porcine pseudorabies virus double ultrafiltration comprises the following steps:
step 1: assembling the system, and then carrying out online cleaning and online sterilization on the system;
step 2: adding porcine pseudorabies virus into an antigen mixing tank, and then introducing clean compressed air through a compressed air inlet to provide pressure, so that the porcine pseudorabies virus sequentially passes through a primary clarifying filter and a secondary micro-filter and then enters a first concentration tank;
step 3: pumping the porcine pseudorabies virus into a heavy ultrafilter by using a first peristaltic pump, and collecting the liquid at the penetrating end to a second concentration tank until the porcine pseudorabies virus liquid in the first concentration tank completely penetrates through the heavy ultrafilter;
step 4: adding PBS solution with the same volume as that of porcine pseudorabies virus liquid into a first concentration tank, and continuously collecting the liquid at the penetrating end into a second concentration tank by using a first peristaltic pump;
step 5: starting a second peristaltic pump to enable the liquid in the second concentration tank to pass through a double ultrafilter, adding PBS solution into the second concentration tank after the liquid in the second concentration tank reaches the required volume, enabling the total volume of the liquid in the second concentration tank to be the same as the initial volume, and then continuing concentrating; repeatedly washing and filtering for many times until the process requirement is met; the liquid passing through the end is waste liquid, and is discarded after inactivation;
step 6: the virus liquid in the second concentration tank was resuspended using PBS for subsequent seeding, sub-packaging, and lyophilization.
Further, the step 1 specifically includes: installing the system in hundred-grade environment, and then checking the air tightness of the system by introducing clean compressed air; then pumping the alkaline solution in an alkaline tank into a system for alkaline water washing, adding purified water into a PBS storage tank, pumping the purified water into the system for purified water dealkalization, adding PBS solution into the PBS storage tank, washing by using the PBS solution, and then purging by using clean compressed air to realize online cleaning and sterilization of the system; after sterilization, clean compressed air is introduced to maintain the system pressure at 0.1MPa, and aseptic maintenance is performed.
Further, the primary ultra-filter and the secondary ultra-filter are sterilized by using alkali liquor circulation for 0.5-1h, then are washed to be neutral by using purified water, and finally are discharged by using PBS solution circulation and balance for 1-2 h.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the invention, aseptic control is performed through online cleaning and sterilization, clarification filtration and microfiltration are performed through filter elements with two apertures, and double ultrafiltration is performed through tangential flow membrane bags with two apertures, so that the purification of the porcine pseudorabies virus is realized, and the problems of high cost and the like of the conventional virus purification centrifugation, dead-end filtration and chromatography technology are overcome;
(2) The method of the invention can greatly reduce the production cost, realize aseptic control and remarkably improve the yield and quality of the vaccine.
Drawings
FIG. 1 is a schematic diagram of a porcine pseudorabies virus dual ultrafiltration system of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Example 1
A porcine pseudorabies virus dual ultrafiltration system comprises an antigen mixing tank 2, a first concentration tank 3 and a second concentration tank 5 which are connected in sequence;
a primary clarifying filter 9 and a secondary microfilter 10 are arranged between the antigen mixing tank 2 and the first concentrating tank 3;
a heavy ultrafilter 4 is arranged between the first concentrating tank 3 and the second concentrating tank 5; the outlet of the second concentration tank 5 is provided with a double ultrafilter 7;
the porcine pseudorabies virus dual ultrafiltration system further comprises a PBS storage tank 1 and an alkali tank 6 which are communicated with the antigen mixing tank 2, the first concentration tank 3 and the second concentration tank 5, and the PBS storage tank and the alkali tank are used for carrying out online cleaning and sterilization on the system.
According to the invention, aseptic control is performed through online cleaning and sterilization, clarification filtration and microfiltration are performed through filter elements with two apertures, and double ultrafiltration is performed through tangential flow membrane bags with two apertures, so that the purification of the porcine pseudorabies virus is realized, and the problems of high cost and the like of the conventional virus purification centrifugation, dead-end filtration and chromatography technology are overcome.
Further, the outlet of the PBS storage tank 1 is provided with a sterilizing filter element 8, and then is communicated with the first concentration tank 3, the second concentration tank 5, the primary ultra-filter 4, the dual ultra-filter 7 and the alkali tank 6 through a primary clarification filter 9 and a secondary micro-filter 10 in a pipeline manner, so that the online cleaning and sterilizing are facilitated.
Preferably, the primary clarifying filter 9 is a filter element with a pore size of 50 μm; the secondary micro-filter 10 is a filter element with the aperture of 0.45 μm; the heavy ultrafilter 4 is a tangential flow ultrafiltration membrane bag with the aperture of 0.2 mu m; the double ultrafilter 7 is a tangential flow ultrafiltration membrane bag with a 300kDa aperture, and different filter apertures are set, so that cell fragments, macromolecular protein polymers, micromolecular hybrid proteins and the like in the virus liquid can be effectively filtered out, and a better purification effect is obtained.
Further, compressed air inlets are formed in the tops of the PBS storage tank 1, the antigen mixing tank 2, the first concentration tank 3, the second concentration tank 5 and the alkali tank 6, and an air filter 11 and a valve 12 are arranged above the air inlets and used for filtering compressed air and controlling air on-off; the first concentrating tank 3 is also communicated with a first peristaltic pump (not shown); the second concentrating tank 5 is also connected to a second peristaltic pump (not shown) and clean air is fed through a compressed air inlet for drying and for providing circulating power to the system.
The top parts of the PBS storage tank 1, the antigen mixing tank 2, the first concentration tank 3, the second concentration tank 5, the alkali tank 6, the sterilizing filter element 8, the primary clarifying filter 9 and the secondary micro-filter 10 are respectively provided with a pressure gauge 13 for detecting the internal pressure of the system in real time.
Example 2
A method for purifying porcine pseudorabies virus by double ultrafiltration, comprising the following steps:
step 1: assembling the system, and then performing online cleaning and online sterilization on the system
Installing the system in hundred-grade environment, and then checking the air tightness of the system by introducing clean compressed air; then pumping 0.1M sodium hydroxide alkali solution in an alkali tank into a system for alkali water flushing, adding purified water into a PBS storage tank, pumping the purified water into the system for alkali removal, adding 0.1M PBS solution into the PBS storage tank, flushing with the 0.1M PBS solution, and then purging with clean compressed air to realize online cleaning and sterilization of the system; circulating a heavy ultrafilter and a double ultrafilter with 0.1M sodium hydroxide lye for 0.5-1h for sterilization, then flushing with purified water to the center, and finally circulating and balancing with 0.1M PBS solution for 1-2h for discharging; after sterilization, clean compressed air is introduced to maintain the pressure of the system at 0.1MPa, and aseptic maintenance is carried out;
step 2: adding 10L of porcine pseudorabies virus into an antigen mixing tank, and then introducing clean compressed air through a compressed air inlet to provide pressure, so that the porcine pseudorabies virus sequentially passes through a primary clarifying filter and a secondary microfilter, and then enters a first concentration tank;
step 3: pumping the porcine pseudorabies virus into a heavy ultrafilter by using a first peristaltic pump, and collecting the liquid at the penetrating end to a second concentration tank until the porcine pseudorabies virus liquid in the first concentration tank completely penetrates through the heavy ultrafilter;
step 4: adding 10L PBS solution with the same volume as the porcine pseudorabies virus liquid into a first concentration tank, and continuously collecting the liquid passing through the end into a second concentration tank by using a first peristaltic pump;
step 5: starting a second peristaltic pump to enable the liquid in the second concentration tank to pass through a double ultrafilter, adding 15L PBS solution into the second concentration tank after the liquid in the second concentration tank reaches 5L, enabling the total volume of the liquid in the second concentration tank to be the same as the initial volume, and then continuing concentrating; repeatedly washing and filtering for 3-5 times; the liquid passing through the end is waste liquid, and is discarded after inactivation;
step 6: the virus liquid in the second concentration tank was resuspended using PBS for subsequent seeding, sub-packaging, and lyophilization.
The samples were tested with the BCA protein concentration kit, with a residual rate of 5.7-6.1% soluble protein, and a lower residual rate, demonstrating that most was removed.
In conclusion, the system provided by the invention has reasonable design and good purification effect, and can obviously improve the mixing amount and quality of the vaccine.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (7)
1. The porcine pseudorabies virus double ultrafiltration device is characterized by comprising an antigen mixing tank, a first concentration tank and a second concentration tank which are connected in sequence;
a primary clarifying filter and a secondary microfilter are arranged between the antigen mixing tank and the first concentrating tank;
a heavy ultrafilter is arranged between the first concentrating tank and the second concentrating tank; the outlet of the second concentration tank is provided with a double ultrafilter;
the porcine pseudorabies virus dual ultrafiltration device further comprises a PBS storage tank and an alkali tank which are communicated with the antigen mixing tank, the first concentration tank and the second concentration tank, and the PBS storage tank and the alkali tank are used for cleaning and sterilizing the device on line;
the primary clarifying filter is a filter element with the aperture of 50 mu m;
the secondary micro-filter is a filter element with the aperture of 0.45 mu m;
the one-weight ultrafilter is a tangential flow ultrafiltration membrane bag with the aperture of 0.2 mu m;
the double ultrafilter is a tangential flow ultrafiltration membrane bag with a 300kDa aperture;
the PBS storage tank, the antigen mixing tank, the first concentration tank, the second concentration tank and the top of the alkali tank are all provided with compressed air inlets.
2. The porcine pseudorabies virus dual ultrafiltration device according to claim 1, wherein a sterilizing filter element is arranged at the outlet of the PBS storage tank, and then is communicated with the first concentration tank, the second concentration tank, the one-weight ultrafilter, the two-weight ultrafilter and the alkali tank through a primary clarification filter and a secondary microfilter.
3. The porcine pseudorabies virus dual ultrafiltration device according to claim 2, wherein the first concentration tank is further communicated with a first peristaltic pump;
the second concentration tank is also communicated with a second peristaltic pump.
4. A method for purifying porcine pseudorabies virus by double ultrafiltration, which is characterized by adopting the porcine pseudorabies virus double ultrafiltration device of claim 3.
5. The method for purifying porcine pseudorabies virus double ultrafiltration according to claim 4, comprising:
step 1: assembling the device, and then carrying out online cleaning and online sterilization on the device;
step 2: adding the porcine pseudorabies virus into an antigen mixing tank, and continuously introducing clean compressed air through a compressed air inlet, so that the porcine pseudorabies virus sequentially passes through a primary clarifying filter and a secondary micro-filter and enters a first concentration tank;
step 3: pumping the porcine pseudorabies virus into a heavy ultrafilter by using a first peristaltic pump, and collecting the liquid at the penetrating end to a second concentration tank until the porcine pseudorabies virus liquid in the first concentration tank completely penetrates through the heavy ultrafilter;
step 4: adding PBS solution with the same volume as that of porcine pseudorabies virus liquid into a first concentration tank, and continuously collecting the liquid at the penetrating end into a second concentration tank by using a first peristaltic pump;
step 5: starting a second peristaltic pump to enable the liquid in the second concentration tank to pass through a double ultrafilter, adding PBS solution into the second concentration tank after the liquid in the second concentration tank reaches the required volume, enabling the total volume of the liquid in the second concentration tank to be the same as the initial volume, and then continuing concentrating; repeatedly washing and filtering for many times until the process requirement is met; the liquid passing through the end is waste liquid, and is discarded after inactivation;
step 6: and (3) re-suspending the virus liquid in the second concentration tank by using PBS solution for subsequent seedling preparation, split charging and freeze-drying.
6. The method for purifying porcine pseudorabies virus according to claim 5, wherein the step 1 specifically comprises: in hundred-grade environment, installing a device, and checking the air tightness of the device by introducing clean compressed air; then pumping the alkali solution in the alkali tank into a device for alkali solution flushing; adding purified water into a PBS storage tank, pumping the purified water into a device for dealkalization, adding PBS solution into the PBS storage tank, flushing by using the PBS solution, and then purging by using clean compressed air to realize online cleaning and sterilization of the device; after sterilization, clean compressed air is introduced to maintain the pressure of the device at 0.1MPa, and aseptic maintenance is performed.
7. The method for purifying porcine pseudorabies virus according to claim 6, wherein the one-time ultrafilter and the two-time ultrafilter are sterilized by circulating alkali liquor for 0.5-1h, then are washed to be neutral by purified water, and finally are discharged by circulating and balancing PBS solution for 1-2 h.
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| CN116200346B (en) * | 2023-05-05 | 2023-09-12 | 北京赛尔富森生物科技有限公司 | Method and system for virus single-pass membrane ultrafiltration concentration |
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| CN110129287A (en) | 2019-08-16 |
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Address after: No. 85 Keyun Road, High tech Zone, Qingdao, Shandong Province, 266000 Patentee after: Qingdao Blue Animal Health Group Co.,Ltd. Patentee after: QINGDAO VLAND BIOTECH Inc. Address before: No. 16 Tianhai Road, Hongdao Street, Hongdao Economic Zone, Qingdao City, Shandong Province, 266000 Patentee before: QINGDAO VLAND BIOTECH GROUP CO.,LTD. Patentee before: QINGDAO VLAND BIOTECH Inc. |