WO2018103619A1 - 一种口蹄疫疫苗的制备方法 - Google Patents

一种口蹄疫疫苗的制备方法 Download PDF

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Publication number
WO2018103619A1
WO2018103619A1 PCT/CN2017/114553 CN2017114553W WO2018103619A1 WO 2018103619 A1 WO2018103619 A1 WO 2018103619A1 CN 2017114553 W CN2017114553 W CN 2017114553W WO 2018103619 A1 WO2018103619 A1 WO 2018103619A1
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Prior art keywords
foot
membrane
mouth disease
microfiltration
ultrafiltration
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PCT/CN2017/114553
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English (en)
French (fr)
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马贵军
张震
聂东升
罗盘棋
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申联生物医药（上海）股份有限公司
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Priority to GB1908107.4A priority Critical patent/GB2572275B/en
Priority to US16/467,058 priority patent/US11013794B2/en
Publication of WO2018103619A1 publication Critical patent/WO2018103619A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D36/00—Filter circuits or combinations of filters with other separating devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32151—Methods of production or purification of viral material
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32161—Methods of inactivation or attenuation
    • C12N2770/32163—Methods of inactivation or attenuation by chemical treatment
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32171—Demonstrated in vivo effect

Definitions

  • the present application relates to the field of veterinary biological products, and in particular, the present invention relates to a method for preparing a foot-and-mouth disease vaccine, a foot-and-mouth disease vaccine prepared by the method, and the use of the foot-and-mouth disease vaccine for preparing a medicament for preventing foot-and-mouth disease in animals. And a device for preparing a foot-and-mouth disease vaccine.
  • Foot and Mouth Disease is an acute, febrile, highly contagious zoonotic disease caused by foot-and-mouth disease virus.
  • Foot-and-mouth disease mainly affects cloven-hoofed animals, and its clinical diagnosis is characterized by blisters in the oral mucosa, hooves and breast skin.
  • a variety of foot-and-mouth disease vaccines for controlling and preventing foot-and-mouth disease have been developed.
  • For the preparation of foot-and-mouth disease vaccines a simple combination of various separation and purification processing units is currently used in the art, for example, continuous flow centrifugation with DC filtration, depth filtration, or hollow fiber filtration, PEG precipitation or chromatography.
  • the harvested foot-and-mouth disease virus cell culture solution is separated and purified to obtain a purified foot-and-mouth disease virus vaccine.
  • the present application relates to a method for preparing a foot-and-mouth disease vaccine, a foot-and-mouth disease vaccine prepared by the method, a use of the foot-and-mouth disease vaccine for preparing a medicament for preventing foot-and-mouth disease in animals, and a device for preparing a foot-and-mouth disease vaccine.
  • the present application provides a method for preparing a foot-and-mouth disease vaccine, comprising the steps of: (i) obtaining a foot-and-mouth disease virus cell culture solution; and (ii) passing the foot-and-mouth disease virus cell culture solution through a double membrane integrated filtration system.
  • the dual membrane integrated filtration system comprises a microfiltration device and an ultrafiltration device arranged in parallel, the microfiltration device comprising a microfiltration liquid tank, a first main pump and a microfiltration membrane, the ultrafiltration
  • the apparatus comprises a concentration tank, a second main pump and an ultrafiltration membrane; wherein the microfiltration apparatus is for removing large particle impurities in the foot-and-mouth disease virus cell culture solution, and the ultrafiltration device is used for removing the foot-and-mouth disease virus cell culture a small molecular impurity in the liquid; the microfiltration device and the ultrafiltration device simultaneously operate to simultaneously perform microfiltration and ultrafiltration on the foot-and-mouth disease virus cell culture solution, thereby forming a concentrate containing the foot-and-mouth disease virus in the concentration tank (iii) collecting the concentrate containing the foot-and-mouth disease virus obtained in the step (ii).
  • the volume of the concentration tank is equal to a target concentration volume of the foot-and-mouth disease virus cell culture fluid or no more than 1.5 times the target concentration volume. In certain embodiments, the volume of the concentration tank is from 1/5 to 1/50 of the initial volume of the foot-and-mouth disease virus cell culture medium.
  • the microfiltration membrane has a pore size of from 0.1 ⁇ m to 0.45 ⁇ m.
  • the ultrafiltration membrane is a hollow fiber ultrafiltration membrane having a pore size of from 100 kD to 500 kD.
  • the first washing filtrate is further added to the microfiltration liquid tank to pass the double membrane.
  • An integrated filtration system to obtain a concentrate.
  • the first wash filtrate is a buffer solution having a pH of 7.2 to pH 9 and a conductivity of 5 to 300 mS/cm.
  • the volume of the first wash filtrate added is from 1 to 5 times the volume of the microfiltration retentate.
  • the ultrafiltration filtrate obtained in the step (ii) is added as a first wash filtrate to the microfiltration liquid tank, and the ultrafiltration filtrate is passed through the double Membrane is combined with an integrated filtration system to obtain a concentrate.
  • the second washing filtrate is added to the concentrated solution containing the foot-and-mouth disease virus obtained in the step (ii), and the small molecular impurities in the concentrated solution are passed through the ultrafiltration membrane to obtain a purified virus concentrated. liquid.
  • the second wash filtrate is a buffer solution having a pH of 7.2 to pH 9 and a conductivity of 5 to 300 mS/cm.
  • the volume of the second wash filtrate added is from 1 to 10 times the volume of the virus concentrate.
  • the microfiltration device is coupled to a first constant flow pump, and the ultrafiltration device is coupled to a second constant flow pump to dynamically control per-membrane flux.
  • the first constant flow pump is disposed at a filtration end of the microfiltration device
  • the second constant current pump is disposed at a filtration end of the ultrafiltration device.
  • the membrane flux is from 65% to 100% of the critical membrane flux of the microfiltration membrane or ultrafiltration membrane. In certain embodiments, the membrane flux is 10-150 LMH.
  • the microfiltration membrane and the ultrafiltration membrane each have a shear rate of 1500 to 4000 s -1 . In some embodiments, the microfiltration membrane and the ultrafiltration membrane each have a material to membrane area ratio of from 10 to 500 L/m 2 .
  • the concentrate containing foot-and-mouth disease virus is inactivated after step (iii). In certain embodiments, the concentrate containing foot-and-mouth disease virus is emulsified after the inactivation. In certain embodiments, the concentrate containing foot-and-mouth disease virus is further purified before or after the inactivation. In certain embodiments, the further purification is performed by PEG precipitation or chromatography. In certain embodiments, the chromatographic methods include exclusion chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.
  • the microfiltration device and the ultrafiltration device are cleaned and regenerated after step (iii).
  • the cleaning regeneration comprises the steps of: (a) separately adding pure water to the microfiltration device and the ultrafiltration device to separately clean the microfiltration membrane and the ultrafiltration membrane therein, during the cleaning process.
  • the membrane shear rate is increased to 8000 s -1 to 16000 s -1 , the membrane flux is controlled at 10 to 500 LMH; (b) after the step (a) is washed, 0.1 to 0.5 M NaOH solution is separately added to the microfiltration device and the chamber.
  • the ultrafiltration device is further cleaned, the solution temperature is 45-55 ° C, the membrane shear rate is 8000 s -1 to 16000 s -1 , the membrane flux is controlled at 50-500 LMH, and the time is 30-60 minutes; (c) After the step (b), the microfiltration device and the ultrafiltration device are separately rinsed with pure water until the pH of the microfiltration filtrate and the ultrafiltration filtrate is reduced to below 9.
  • the foot-and-mouth disease virus strain comprises one or more foot-and-mouth disease virus serotype strains.
  • the serotype is O, A, C, SAT1, SAT2, SAT3 or Asia1.
  • the present application relates to a foot-and-mouth disease vaccine prepared according to the methods described herein.
  • the present application relates to the use of a foot-and-mouth disease vaccine according to the present application for the preparation of a medicament for preventing foot-and-mouth disease in an animal.
  • the present application is directed to a method of preventing foot-and-mouth disease in an animal comprising administering to the animal an immunologically effective amount of a foot-and-mouth disease vaccine according to the present application.
  • the present application relates to a foot-and-mouth disease vaccine according to the present application for use in preventing foot-and-mouth disease in an animal.
  • the present application relates to an apparatus for preparing a foot-and-mouth disease vaccine comprising a dual membrane integrated filtration system comprising a microfiltration device and an ultrafiltration device disposed in parallel,
  • the microfiltration device comprises a microfiltration liquid tank, a first main pump and a microfiltration membrane, the ultrafiltration device comprising a concentration tank, a second main pump and An ultrafiltration membrane; wherein the microfiltration device is for removing large particle impurities in the foot-and-mouth disease virus cell culture solution, and the ultrafiltration device is for removing small molecular impurities in the foot-and-mouth disease virus cell culture solution;
  • the filter device and the ultrafiltration device are simultaneously operated to simultaneously perform microfiltration and ultrafiltration on the foot-and-mouth disease virus cell culture solution, thereby forming a concentrate containing the foot-and-mouth disease virus in the concentration tank.
  • the microfiltration device and the ultrafiltration device are interconnected such that both the microfiltration filtrate and the ultrafiltration retentate are placed in the concentration tank to form the concentrate containing the foot-and-mouth disease virus. .
  • the dual membrane integrated filtration system further includes a first constant flow pump and a second constant flow pump, the microfiltration device being coupled to the first constant flow pump, the ultrafiltration device Connected to the second constant current pump to dynamically control the membrane flux.
  • the first constant flow pump is disposed at a filtration end of the microfiltration device
  • the second constant current pump is disposed at a filtration end of the ultrafiltration device.
  • Fig. 1 is a schematic view showing the preparation process of the foot-and-mouth disease vaccine of Example 1 of the present application.
  • Fig. 2 is a schematic view showing the preparation process of the foot-and-mouth disease vaccine of Example 2 of the present application.
  • Fig. 3 is a view showing the SDS-PAGE electrophoresis pattern of the foot-and-mouth disease virus antigen of Example 1 of the present application.
  • Fig. 4 shows the results of HPLC detection of the foot-and-mouth disease virus antigen of Example 1 of the present application.
  • Fig. 5 is a graph showing the water flux measurement values of the washing recovery after the continuous use of the microfiltration membrane and the ultrafiltration membrane of Example 5 of the present application.
  • the present application provides a method for preparing a foot-and-mouth disease vaccine, comprising the steps of: (i) obtaining a foot-and-mouth disease virus cell culture solution; (ii) separating the foot-and-mouth disease virus cell culture solution through a double membrane integrated filtration system.
  • the dual membrane integrated filtration system comprises a microfiltration device and an ultrafiltration device arranged in parallel,
  • the microfiltration device comprises a microfiltration solution tank, a first main pump and a microfiltration membrane, the ultrafiltration device comprising a concentration tank, a second main pump and an ultrafiltration membrane; wherein the microfiltration device is for removing the foot and mouth disease a large particle impurity in the virus cell culture solution, the ultrafiltration device is for removing small molecular impurities in the foot-and-mouth disease virus cell culture solution; the microfiltration device and the ultrafiltration device simultaneously operate on the foot-and-mouth disease virus cell
  • the culture solution is simultaneously subjected to microfiltration and ultrafiltration to form a concentrate containing the foot-and-mouth disease virus in the concentration tank; (iii) collecting the concentrated solution containing the foot-and-mouth disease virus obtained in the step (ii).
  • the term "vaccine” as used in this application refers to a composition containing one or more antigens having immunity to the body. After introduction of the vaccine into a host animal, the vaccine is capable of stimulating the host animal to produce an immune response against the one or more antigens.
  • the term "host animal” as used in this application refers to an animal that is capable of being invaded by foot-and-mouth disease virus and allowing the foot-and-mouth disease virus to replicate in its body.
  • the host animal is a cloven-hoofed animal.
  • the host animal is a domestic animal such as a pig, cow, or sheep.
  • the host animal is a pig. Invasion and replication of foot-and-mouth disease virus in host animals may cause the host animal to develop clinical symptoms of foot-and-mouth disease, or may not cause any clinical symptoms in the host animal.
  • the foot-and-mouth disease virus described in the present application may include one or more strains of foot-and-mouth disease virus serotype.
  • the serotype is O, A, C, SAT1, SAT2, SAT3 or Asia1. Each main type is subdivided into several subtypes, and more than 70 subtypes have been discovered.
  • the serotype is Form A.
  • the method for preparing a foot-and-mouth disease vaccine of the present application is applicable to various strains of foot-and-mouth disease virus serotypes or mixtures thereof.
  • the foot-and-mouth disease virus described in the present application may be a natural foot-and-mouth disease virus obtained by isolation and purification from a natural environment, or may be a recombinant foot-and-mouth disease virus strain obtained by genetic engineering methods, or may be prepared by other expression systems (engineering bacteria, insects, plants). Virroid-like particles, recombinant antigens.
  • a recombinant foot-and-mouth disease virus strain or a foot-and-mouth disease virus fragment can be obtained by a person skilled in the art by genetic engineering methods well known in the art, see, for example, http://www.science.gov/topicpages/v/virus+vaccine+development.html describe.
  • foot-and-mouth disease virus cell culture fluid refers to a cell culture fluid that is infected with foot-and-mouth disease virus and allows replication of the foot-and-mouth disease virus.
  • Foot-and-mouth disease virus cell culture fluids can be prepared using methods well known in the art (see, for example, Yan Daliang et al., Overview of the production process of foot-and-mouth disease inactivated vaccines, Chinese Journal of Veterinary Medicine, 2011 45(1): 41-44).
  • Cells suitable for culturing foot-and-mouth disease virus include milk hamster kidney cells (BHK21 cells), porcine kidney cells (IBRS-2 cells), bovine kidney cells (MDBK cells), African green monkey kidney cells (Vero cells), and the like.
  • BHK21 cells milk hamster kidney cells
  • IBRS-2 cells porcine kidney cells
  • MDBK cells bovine kidney cells
  • Vero cells African green monkey kidney cells
  • the term "dual membrane integrated filtration system” as used in the present application refers to a microfiltration device and an ultrafiltration device which are arranged in parallel, and the microfiltration device and the ultrafiltration device are connected and integrated with each other.
  • the microfiltration device comprises a microfiltration solution tank, a first main pump, and a microfiltration membrane;
  • the ultrafiltration device comprising a concentration tank, a second main pump, and an ultrafiltration membrane, the microfiltration device and the The ultrafiltration device is simultaneously operated to simultaneously perform microfiltration and ultrafiltration on the foot-and-mouth disease virus cell culture liquid, thereby forming a concentrate containing the foot-and-mouth disease virus in the concentration tank.
  • the microfiltration device and the ultrafiltration device are placed side by side and connected to each other such that both the microfiltration filtrate and the ultrafiltration retentate are placed in the concentration tank.
  • the concentrate containing the foot-and-mouth disease virus.
  • microfiltration filtrate refers to a filtrate obtained after the foot-and-mouth disease virus cell culture medium has passed through a microfiltration device.
  • the microfiltration device is for removing large particle impurities in the foot-and-mouth disease virus cell culture solution. After the foot-and-mouth disease virus cell culture solution passes through the microfiltration device, the obtained microfiltration filtrate contains a large particle impurity level and the foot-and-mouth disease before the foot-and-mouth disease virus cell culture solution is passed through the microfiltration device. There is a significant decrease in the level of large particulate impurities in the virus cell culture.
  • the microfiltration device can remove the foot-and-mouth disease virus cell culture solution as compared with the level of large particle impurities in the foot-and-mouth disease virus cell culture solution before passing the foot-and-mouth disease virus cell culture solution through the microfiltration device. At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% of large particulate impurities.
  • the microfiltration device is capable of removing the large particle impurity level in the foot-and-mouth disease virus cell culture fluid prior to passing the foot-and-mouth disease virus cell culture fluid through the microfiltration device.
  • From about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 40% to about 80%, from about 40% to about 90%, about 40% of the foot-and-mouth disease virus cell culture fluid Up to about 100%, from about 50% to about 80%, from about 50% to about 90%, from about 50% to about 100% (eg, from about 60% to about 90%, from about 70% to about 90%), about 60% % to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100% or about 95% to about 100% (for example, about 96% to about 100%, about 97%) % to about 100%, from about 98% to about 100%, from about 99% to about 100%, from about 95% to about 96%, from about 95% to about 97%, from about 95% to about 98% or from about 95% to About 99%) of
  • large particle impurity means an impurity such as cell debris, bacteria, aggregates, flocs, etc. present in the foot-and-mouth disease virus cell culture solution.
  • the smallest diameter of the large particle impurity More than 0.1 ⁇ m.
  • the small molecule impurities and the foot-and-mouth disease virus pass through the microfiltration membrane and are present in the microfiltration filtrate.
  • the microfiltration filtrate is further passed through an ultrafiltration device which can remove small molecular impurities in the foot-and-mouth disease virus cell culture solution, but the foot-and-mouth disease virus cannot pass through the ultrafiltration membrane and is trapped in the ultrafiltration retentate.
  • ultrafiltration retentate refers to a liquid which is intercepted in a concentration tank of an ultrafiltration apparatus which is not passed through an ultrafiltration membrane in the microfiltration filtrate, and is a concentrate containing foot-and-mouth disease virus.
  • the resulting ultrafiltration retentate contains a small molecular impurity level and the microfiltration filtrate before passing the microfiltration filtrate through the ultrafiltration device.
  • the ultrafiltration device can remove the microfiltration filtrate compared to the level of small molecule impurities in the microfiltration filtrate prior to passing the microfiltration filtrate through the ultrafiltration device.
  • the ultrafiltration device is capable of removing the small molecule impurity level in the microfiltration filtrate prior to passing the microfiltration filtrate through the ultrafiltration device. From about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 40% to about 80%, from about 40% to about 90%, about 40% of the microfiltration filtrate.
  • small molecule impurity refers to impurities such as nucleic acid and nucleic acid fragments, host proteins, viral non-structural proteins, protein and virus degradants, and medium components in the foot-and-mouth disease virus cell culture solution.
  • the small molecule impurity has a size of less than 500,000 Daltons. These small molecular impurities are smaller in size than the ultrafiltration membrane and therefore can be present in the ultrafiltration filtrate through the ultrafiltration membrane.
  • ultrafiltration filtrate refers to the filtrate obtained after the microfiltration filtrate has passed through an ultrafiltration unit.
  • the microfiltration liquid tank described in the present application is used for holding the foot-and-mouth disease virus cell culture solution to be separated and purified, and the microfiltration retentate that has not passed through the microfiltration membrane is returned to the microfiltration liquid tank.
  • microfiltration interception as used in this application "Liquid” means a liquid that is trapped in a microfiltration liquid tank without passing through a microfiltration membrane.
  • the microfiltration retentate and the foot-and-mouth disease virus cell culture fluid to be separated and purified are mixed and placed in the In a microfiltration tank, in some embodiments, the microfiltration device of the present application is provided with a stirring device that agitates the microfiltration retentate in the microfiltration tank and to be separated and purified. The foot-and-mouth disease virus cell culture solution was mixed evenly.
  • the concentrating tank described in the present application is used to hold the microfiltration filtrate, and the ultrafiltration retentate that has not passed through the ultrafiltration membrane is also contained in the concentrating tank.
  • the microfiltration filtrate and ultrafiltration retentate are mixed and placed in the concentration tank.
  • the ultrafiltration device of the present application is provided with a stirring device that agitates and mixes the microfiltration filtrate and the ultrafiltration retentate in the concentration tank.
  • the first main pump described in the present application is used for continuously pumping liquid in the microfiltration liquid tank into the microfiltration device
  • the second main pump described in the present application is used for continuously pumping the liquid in the concentration tank into the ultrafiltration.
  • the microfiltration device and the ultrafiltration device are simultaneously operated to simultaneously perform microfiltration and ultrafiltration on the foot-and-mouth disease virus cell culture solution.
  • microfiltration and ultrafiltration are performed simultaneously, that is, at the same time, part of the cell culture solution is filtered through the microfiltration membrane, and some is filtered through the ultrafiltration membrane.
  • the microfiltration filtrate is subjected to ultrafiltration through an ultrafiltration device.
  • the dual membrane integrated filtration system in the present application not only reduces the pipeline connection, reduces the floor space, but also greatly saves the filtration time.
  • the temperature at which microfiltration and ultrafiltration are performed are each 2 to 20 ° C, such as 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C, 8 ° C, 9 ° C, 10 ° C. Any value in the range between any two values of 11 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ° C or above.
  • the temperature at which both microfiltration and ultrafiltration are performed are each 2 to 8 °C. Constant low temperature control helps maintain the stability of the foot-and-mouth disease virus antigenic structure.
  • the volume of the concentration tank is equal to a target concentration volume of the foot-and-mouth disease virus cell culture fluid or no more than 1.5 times the target concentration volume.
  • the volume of the concentration tank is any value within a range between any two values of 1 time, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times or more of the target concentrated volume.
  • the volume of the concentration tank is from 1/5 to 1/50 of the initial volume of the foot-and-mouth disease virus cell culture medium.
  • the volume of the concentration tank is 1/5, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40 of the initial volume of the foot-and-mouth disease virus cell culture solution. Any value within the range between any of the two values of 1/45, 1/50 or above.
  • the microfiltration membrane described in the present application may be a commercially available microfiltration membrane such as a microfiltration hollow fiber membrane series produced by companies such as GE Healthcare Life Sciences, Shibi Pure, and Asahi Kasei Corporation.
  • the microfiltration membrane has a pore diameter of from 0.1 ⁇ m to 0.45 ⁇ m, such as 0.1 ⁇ m, 0.15 ⁇ m, 0.2 ⁇ m, 0.22 ⁇ m, 0.25 ⁇ m, 0.3 ⁇ m, 0.35 ⁇ m, 0.4 ⁇ m, 0.45 ⁇ m or more. Any value between any two numerical ranges.
  • the selection of the microfiltration membrane pore size as small as possible contributes to the recovery of the foot-and-mouth disease virus antigen and the removal of large particle impurities.
  • the ultrafiltration membrane described in the present application may be a commercially available ultrafiltration membrane, such as an ultrafiltration hollow fiber membrane series produced by companies such as GE Healthcare Life Sciences, Shibi Pure, and Asahi Kasei.
  • the ultrafiltration membrane is a hollow fiber ultrafiltration membrane having a pore diameter of 100 kD to 500 kD, for example, any of pore diameters of 100 kD, 150 kD, 200 kD, 250 kD, 300 kD, 350 kD, 400 kD, 450 kD, 500 kD or above.
  • the first washing filtrate is further added to the microfiltration liquid tank to pass the double membrane.
  • An integrated filtration system to obtain a concentrate.
  • "at least a portion” means at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least Any value between 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, even 100% or above any two numerical ranges.
  • the addition of the first washing filtrate facilitates the enrichment of the foot-and-mouth disease virus antigen remaining in the microfiltration liquid tank through the microfiltration membrane into the ultrafiltration device, and the washing of the first washing filtrate can increase the activity of the foot-and-mouth disease virus antigen in the microfiltration process. Recovery rate.
  • the first wash filtrate is a buffer solution having a pH of 7.2 to pH 9 and a conductivity of 5 to 300 mS/cm.
  • the buffer solution is phosphate buffered saline (PBS), borate buffer solution, citrate buffer solution, acetate buffer solution, barbiturate buffer solution or trimethylol Aminomethane buffer (Tris).
  • the buffer solution is a PBS buffer solution.
  • the pH of the buffer solution is any value within a range between 7.2, 7.5, 8.0, 8.5, 9.0, or any two of the above values.
  • the conductivity of the buffer solution is between 5 mS/cm, 50 mS/cm, 100 mS/cm, 150 mS/cm, 200 mS/cm, 250 mS/cm, 300 mS/cm or more. Any value within.
  • the volume of the first wash filtrate added is 1 to 5 times the volume of the microfiltration retentate, Any value within a range between any two values of 1 time, 2 times, 3 times, 4 times, 5 times or more.
  • the amount of the first wash filtrate used may be 1/5, 1/2, 1 or 2 times the initial volume of the cell culture fluid.
  • the ultrafiltration filtrate obtained after microfiltration and ultrafiltration is added as a first washing filtrate to the microfiltration liquid tank, and the foot-and-mouth disease virus antigen retained by the microfiltration membrane is washed and filtered. Enter the ultrafiltration unit to obtain a concentrate.
  • the amount of washing filtrate can be greatly reduced, the cost can be further reduced, and the recovery rate of the foot-and-mouth disease virus antigen can be increased.
  • the second washing filtrate is added to the concentrated solution containing the foot-and-mouth disease virus obtained after microfiltration and ultrafiltration, and further, the small molecular impurities remaining in the concentrated solution are passed through an ultrafiltration membrane to obtain purification. Virus concentrate.
  • the addition of the second washing filtrate facilitates the washing out of small molecular impurities in the ultrafiltration device, and reduces the residual of small molecular impurities in the ultrafiltration retentate, thereby improving the purity of the foot-and-mouth disease virus in the foot-and-mouth disease virus concentrate.
  • the second wash filtrate is a buffer solution having a pH of 7.2 to pH 9 and a conductivity of 5 to 300 mS/cm.
  • the buffer solution is phosphate buffered saline (PBS), borate buffer solution, citrate buffer solution, acetate buffer solution, barbiturate buffer solution or trimethylol Aminomethane buffer (Tris).
  • the buffer solution is a phosphate buffer solution.
  • the pH of the buffer solution is any value within a range between 7.2, 7.5, 8.0, 8.5, 9.0, or any two of the above values.
  • the conductivity of the buffer solution is between 5 mS/cm, 50 mS/cm, 100 mS/cm, 150 mS/cm, 200 mS/cm, 250 mS/cm, 300 mS/cm or more. Any value within.
  • the volume of the second wash filtrate added is 1 to 10 times the volume of the foot-and-mouth disease virus concentrate, for example, 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 Any value in the range between any two values of 8, 8, 9, 10 or more.
  • foot-and-mouth disease virus concentrate or "concentrated solution containing foot-and-mouth disease virus” as used in the present application means that the content of large particle impurities and small molecular impurities is reduced after microfiltration and ultrafiltration treatment, and the ultrafiltration membrane is not passed. The liquid that is trapped in the concentrating tank.
  • the microfiltration device is coupled to a first constant flow pump, and the ultrafiltration device is coupled to a second constant flow pump to dynamically control per-membrane flux.
  • dynamic control refers to maintaining the surface contamination of the microfiltration membrane and ultrafiltration membrane at a temperature that is irreversible and below the threshold by dynamically changing the flux of the membrane. For example, as the concentration factor increases, it needs to be continuously adjusted.
  • the constant current pump reduces the flux of the membrane so that the surface contamination of the membrane is in an irreversible state of contamination, thereby delaying the decrease in the efficiency of the membrane and prolonging the service life of the membrane.
  • pass through refers to the amount of fluid passing through the unit membrane area per unit time.
  • the unit of the membrane flux is expressed by LMH (L/m ⁇ 2*h), that is, one square meter in one hour. The number of fluid volumes of the membrane area.
  • the first constant current pump described in the present application is for controlling the membrane flux of the microfiltration device
  • the second constant current pump is for controlling the membrane flux of the ultrafiltration device.
  • the first constant current pump is disposed at a filtration end of the microfiltration device
  • the second constant current pump is disposed at a filtration end of the ultrafiltration device to achieve a membrane crossing Dynamic control of flux.
  • the membrane flux can be controlled by controlling the concentration factor of the foot-and-mouth disease virus cell culture medium (i.e., the volume obtained by dividing the volume of the cell culture medium by the initial volume of the cell culture medium).
  • concentration factor i.e., 1/5 to 1/50
  • the membrane flux can maintain a reasonable range that can be operated. Otherwise, the concentration factor is too high, which may cause the membrane flux to be too low, and the operation time is too long; A low concentration factor will result in low recovery of the foot-and-mouth disease virus antigen, which requires washing with a large amount of washing filtrate.
  • the membrane flux is below a critical membrane flux of the microfiltration membrane or ultrafiltration membrane.
  • critical membrane flux as used in this application is a threshold for reversible contamination and irreversible contamination of a microfiltration membrane or ultrafiltration membrane.
  • the critical membrane flux varies with the pore size, material, and structure of the membrane, and the sample state and operating conditions also affect the critical membrane flux.
  • the critical membrane flux of the membrane can be determined by a variety of methods known in the art, such as by increasing the membrane flux by gradient to increase the membrane pressure, or by increasing the membrane pressure by gradient to detect membrane flux ( See, for example, RW Field et al., Critical flux concept for microfiltration fouling, Journal of Membrane Science, 1995, 100(3): 259-272).
  • membrane flux is one of the important factors affecting the productivity and membrane life of foot-and-mouth disease virus. If the membrane flux exceeds the critical membrane flux, it may cause irreversible contamination of the membrane, thereby reducing the membrane life; if the membrane flux is too low than the critical membrane flux, it may lead to prolonged production time, production The stability of the system is maintained at an increased cost, resulting in low production efficiency. Therefore, it is necessary to select a suitable membrane flux below the critical membrane flux to ensure the high yield of the foot-and-mouth disease virus and the removal of impurities, as well as to ensure the stability of the system control and maintain a long service life, thereby greatly reducing production. cost.
  • the membrane flux is between 65% and 100% of the critical membrane flux of the microfiltration membrane or ultrafiltration membrane, for example, the membrane flux is a critical membrane of the microfiltration membrane or ultrafiltration membrane. 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the flux Or any value within the range between any two of the above values.
  • the membrane flux is 10-150 LMH, for example, 10 LMH, 20 LMH, 30 LMH, 40 LMH, 50 LMH, 60 LMH, 70 LMH, 80 LMH, 90 LMH, 100 LMH, 110 LMH, 120 LMH, 130 LMH, 140 LMH, 150 LMH, or any two of the above. Any value within the range between values.
  • the shear rate of the fluid as it passes through the microfiltration membrane and the ultrafiltration membrane is also one of the important factors affecting the productivity and membrane life of the foot-and-mouth disease virus. If the excessive shear rate is used, it will cause shear damage and dissociation of the virus due to the shear tolerance range of the foot-and-mouth disease virus antigen, which will reduce the yield; while too small a shear rate will reduce the membrane flux and reduce the membrane. The efficiency of separation.
  • the microfiltration membrane and the ultrafiltration membrane each have a shear rate of 1500-4000 s -1 , and the shear rate can result in a loss of the foot-and-mouth disease virus antigen of no more than 5%.
  • the example of the microfiltration membrane and ultrafiltration membrane are each a shear rate of 1500s -1, 1600s -1, 1700s -1 , 1800s -1, 1900s -1, 2000s -1, 2100s -1, 2200s -1, 2300s -1, 2400s -1, 2500s -1 , 2600s -1, 2700s -1, 2800s -1, 2900s -1, 3000s -1, 3100s -1, 3200s -1, 3300s -1, 3400s -1, 3500s - Any value within the range between 1 and 3600s -1 , 3700s -1 , 3800s -1 , 3900s -1 , 4000s -1 or above.
  • the material to membrane area ratio of the microfiltration membrane and the ultrafiltration membrane is also one of the important factors affecting the yield and membrane life.
  • the material to membrane area ratio is calculated by dividing the volume of the liquid to be treated by the membrane area. If the material to membrane area ratio is too high, the membrane loading may be exceeded, the critical membrane flux and its operating conditions are difficult to maintain, resulting in irreversible membrane fouling, or too long operating time, low production efficiency; if material and membrane area When the ratio is too low, the film efficiency is not fully exerted, and the production cost is increased. Therefore, it is necessary to select a suitable material to membrane area ratio, thereby prolonging the service life of the membrane as much as possible while ensuring high yield.
  • the microfiltration membrane and the ultrafiltration membrane have a material to membrane area ratio of 10 to 500 L/m 2 , for example, 10 L/m 2 , 50 L/m 2 , and 100 L/m 2 , 150L/m 2 , 200L/m 2 , 250L/m 2 , 300L/m 2 , 350L/m 2 , 400L/m 2 , 450L/m 2 , 500L/m 2 or more in the range between any two values Any value.
  • the concentrate containing foot-and-mouth disease virus is inactivated after step (iii) above.
  • the concentrate containing the foot-and-mouth disease virus can be inactivated by a method commonly used in the art, for example, the concentrated and purified concentrated solution containing the foot-and-mouth disease virus is inactivated with 1 mM to 3 mM of diethyleneimine (BEI) at 30 ° C for 28 hours, after which Add the blocker sodium thiosulfate in a volume ratio of 1.6%, block for 20 min, and reserve at 4 ° C; or add 1 / 4000 ⁇ propiolactone to concentrate and purify the concentrate containing foot-and-mouth disease virus and inactivate it at 37 ° C for 3-4 days. Stored at 4 °C for use (see, for example, Yan Daliang et al., Overview of the production process of foot-and-mouth disease inactivated vaccine, Chinese Journal of Veterinary Medicine, 2011 45(1): 41-44).
  • the concentrate containing foot-and-mouth disease virus is emulsified after the inactivation.
  • the concentrate containing the foot-and-mouth disease virus can be emulsified by a method commonly used in the art, for example, the aqueous phase is prepared first, that is, the concentrated solution containing the foot-and-mouth disease virus is diluted with the sterilized physiological saline, and the oil phase is prepared.
  • the oil phase adjuvant such as Montanide ISA 206
  • the oil phase adjuvant is sterilized at 120 ° C for use; then the aqueous phase and the oil phase are preheated to 30 ° C, and the ratio is 1:1 by weight, in the case of low speed agitation,
  • the aqueous phase preheated to 30 ° C is slowly added to the oil phase which is also preheated to 30 ° C, emulsified for about 20 minutes, the aqueous phase and the oil phase are thoroughly mixed, and emulsified into a two-way (W/O/W) oil emulsion vaccine.
  • W/O/W two-way
  • the concentrate containing foot-and-mouth disease virus is further purified before or after the inactivation.
  • the further purification is carried out by PEG (polyethylene glycol) precipitation or chromatography.
  • the PEG precipitation method is a technique for separating the foot-and-mouth disease virus and other impurities in the concentrate by adding PEG to change the physical and chemical parameters of the concentrate containing the foot-and-mouth disease virus, controlling the solubility of various components in the concentrate.
  • Commonly used precipitants are PEG2000, PEG4000, PEG6000, and the like.
  • the general procedure includes adding PEG to the concentrate containing the foot-and-mouth disease virus to form a precipitate, allowing the resulting precipitate to stand with the mother liquor for a period of time to promote the formation of precipitated particles, and then centrifuging or filtering to collect the precipitate.
  • the chromatographic methods include exclusion chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.
  • Exclusion Chromatography also known as gel chromatography, is the difference in the molecular weight of foot-and-mouth disease virus and other impurities in the concentrate containing foot-and-mouth disease virus and the degree of penetration on the filler to make the foot-and-mouth disease virus and other impurities Separation.
  • Fillers for gel chromatography include polyacrylamide gels, crosslinked dextran gels, agarose gels, polyphenylene gels, silica gels, polymethacrylates, and the like.
  • Various gel chromatography packings are also commercially available, such as gel chromatography packings produced by GE Healthcare Life Sciences, Tosho, Merck Millipore, Boglon, Namicrotech, Xi'an Lanxiao, and the like.
  • the gel chromatography packing employed is a cross-linked dextran gel. Further purification of the concentrate containing foot-and-mouth disease virus by gel chromatography is generally carried out by adding a gel chromatography packing to a concentrate containing foot-and-mouth disease virus, and then loading the packing into a chromatographic column for chromatographic manipulation, or containing foot and mouth disease The concentrate of the virus is directly added to a column packed with a gel chromatography cartridge for chromatography.
  • Ion exchange chromatography uses the interaction of charged groups on the ion exchange chromatography packing with different charged components in the concentrated solution containing foot-and-mouth disease virus to separate the foot-and-mouth disease virus from other impurities, thereby further purifying the foot-and-mouth disease virus. According to the charge on the surface of the ion exchange chromatography packing, it can be divided into cation exchange color. Spectral and anion exchange chromatography.
  • the ion exchange groups on the surface of the ion exchange chromatography packing employed are diethylaminoethyl (DEAE), quaternary ammonium (Q), diethylaminopropyl (ANX), carboxymethyl ( CM), sulfonic acid group (SP), and the like.
  • Various ion exchange chromatography packings are also commercially available, such as ion exchange chromatography packings produced by GE Healthcare Life Sciences, Tosho, Merck Millipore, Boglon, Nanotech, Xi'an Lanxiao, and the like.
  • Further purification of the concentrate containing foot-and-mouth disease virus by ion exchange chromatography is generally carried out by adding the ion exchange chromatography packing to the concentrate containing the foot-and-mouth disease virus, and then loading the packing into the column for chromatography, or it will contain foot-and-mouth disease.
  • the virus concentrate is added directly to the column containing the ion chromatography packing for chromatography.
  • Hydrophobic chromatography is the interaction between the hydrophobic group on the surface of the foot-and-mouth disease virus and the hydrophobic group on the surface of the foot-and-mouth disease virus in the hydrophobic interaction chromatography to achieve the adsorption of the foot-and-mouth disease virus on the chromatographic packing. Phase separation of impurities with weak adsorption and adsorption does not occur.
  • the hydrophobic group on the surface of the hydrophobic chromatography packing employed is butyl, butylthio, phenyl, or octyl.
  • hydrophobic chromatography fillers are also commercially available, such as hydrophobic chromatography packings produced by GE Healthcare Life Sciences, Tosho, Merck Millipore, Boglon, Nanotech, Xi'an Lanxiao, and the like. Further purification of the concentrate containing foot-and-mouth disease virus by hydrophobic chromatography is generally carried out by adding a hydrophobic interaction chromatography cartridge to a concentrate containing foot-and-mouth disease virus, and then loading the filler into the column for chromatography or containing The concentrate of foot-and-mouth disease virus is directly added to a column packed with a hydrophobic interaction chromatography column for chromatography.
  • Affinity chromatography is to put a specific structure of the affinity molecule filler into a solid phase adsorbent and place it in a chromatography column.
  • the concentrate containing the foot-and-mouth disease virus passes through the column, it has affinity with the adsorbent.
  • the foot-and-mouth disease virus is adsorbed and retained in the column, and the impurities without affinity are directly discharged due to not being adsorbed, thereby realizing the separation of the foot-and-mouth disease virus from the impurities.
  • the appropriate eluent is used to change the binding conditions to elute the bound foot-and-mouth disease virus.
  • the adsorbent used is alumina, silica gel, polyamide, and the like.
  • affinity molecule fillers are also commercially available, such as heparin affinity media. Further purification of the concentrate containing foot-and-mouth disease virus by affinity chromatography is generally carried out by adding the affinity molecule filler to the concentrate containing the foot-and-mouth disease virus, and then loading the filler into the column for chromatography, or The concentrate containing the foot-and-mouth disease virus is directly added to a column packed with an affinity molecule for chromatography.
  • the microfiltration device and the ultrafiltration device are cleaned and regenerated after step (iii) above.
  • the microfiltration membrane in the microfiltration device and the ultrafiltration membrane in the ultrafiltration device cause membrane during long-term operation
  • the amount of water permeation decreases as the run time increases, ie membrane fouling occurs. Therefore, it is necessary to clean and regenerate the microfiltration membrane and the ultrafiltration membrane, restore the performance of the membrane, and prolong the service life of the membrane, thereby reducing the production cost.
  • the cleaning regeneration comprises the steps of: (a) separately adding pure water to the microfiltration device and the ultrafiltration device to separately clean the microfiltration membrane and the ultrafiltration membrane therein, during the cleaning process.
  • the membrane shear rate is increased to 8000 s -1 to 16000 s -1 , the membrane flux is controlled at 10 to 500 LMH; (b) after the step (a) is washed, 0.1 to 0.5 M NaOH solution is separately added to the microfiltration device and the chamber.
  • the ultrafiltration device is further cleaned, the solution temperature is 45-55 ° C, the membrane shear rate is 8000 s -1 to 16000 s -1 , the membrane flux is controlled at 50-500 LMH, and the time is 30-60 minutes; (c) After the step (b), the microfiltration device and the ultrafiltration device are separately rinsed with pure water until the pH of the microfiltration filtrate and the ultrafiltration filtrate is reduced to below 9.
  • the step (a) up to a shear rate of membrane 8000s -1, 9000s -1, 10000s -1 , 11000s -1, 12000s -1, 13000s -1, 14000s -1, 15000s - 1 , 16000 s -1 or any value within the range between any two values the membrane flux is controlled at 10LMH, 50LMH, 100LMH, 150LMH, 200LMH, 250LMH, 300LMH, 350LMH, 400LMH, 450LMH, 500LMH or above Any value within the range between any two values.
  • the temperature of the NaOH solution in the step (b) is 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 ° C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C.
  • membrane shear rate is 8000s -1 , 9000s -1 , 10000s -1 , 11000s -1 , 12000s -1 , 13000s -1 , 14000s -1 , 15000s -1 Any value within the range between any two values of 16000 s -1 or above; the over-membrane flux is controlled at 50 LMH, 100 LMH, 150 LMH, 200 LMH, 250 LMH, 300 LMH, 350 LMH, 400 LMH, 450 LMH, 500 LMH, or both Any value within the range between values; the time is any value within a range between any two values of 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, or more.
  • the pH in step (c) is reduced to any value within a range between any of the two values of 9, 8.5, 8.0, 7.5, 7.0, or above.
  • the membrane flux is still dynamically controlled during the wash regeneration process to prevent contaminants from penetrating the membrane pores at high transmembrane pressure, resulting in difficulty in removal. Moreover, the warm and strong base has a decomposing effect on the contaminants, so that the membrane performance is very well maintained.
  • the present application relates to a foot-and-mouth disease vaccine prepared according to the methods described herein.
  • the foot-and-mouth disease vaccine of the present application can be delivered to an administered animal by an appropriate route, including but not Limited to, by oral route, injection route (such as intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal, etc.), mucosal routes (such as intranasal administration) , oral administration, etc.), sublingual route, rectal route, transdermal route, intraocular route, pulmonary route.
  • the foot-and-mouth disease vaccine of the present application can be administered by an injection route.
  • the amount of the foot-and-mouth disease vaccine varies depending on the activity of the active ingredient, the age and weight of the individual to be administered, and the like.
  • One skilled in the art can readily determine the most appropriate dosage of vaccine based on the aforementioned factors affecting the dosage.
  • the foot-and-mouth disease vaccine prepared according to the method described in the present application can be tested for its immunological potency by various methods known.
  • the prior art provides a variety of test methods for assessing the efficacy of vaccine immunization, for example, challenge protection tests, serological tests, and the like.
  • the challenge test was carried out by artificially infecting the test animals after vaccination with a strong virus, and then determining the immune efficacy of the vaccine based on the incidence of the animals.
  • the challenge test is the most direct method to test the effectiveness of vaccine immunization. Therefore, many countries including China require the use of challenge test to evaluate the immune efficacy of veterinary vaccine.
  • a serological test method can also be used to evaluate the immunological efficacy of the vaccine, for example, by measuring the amount of antibody produced in the serum of the immunized animal by an ELISA method (ie, an enzyme-linked immunosorbent assay) to determine the immunological efficacy of the vaccine.
  • an ELISA method ie, an enzyme-linked immunosorbent assay
  • the antigen (or antibody) When ELISA is used, the antigen (or antibody) is usually bound to a solid phase carrier, and then an antibody (or antigen) is combined with the enzyme to form a conjugate (label), when the conjugate and the solid phase
  • the antigen (or antibody) on the carrier reacts and binds to the corresponding substrate of the enzyme, that is, it undergoes catalytic hydrolysis or redox reaction to form a color, and the color depth generated is proportional to the antigen (or antibody) content to be tested.
  • serological indicators and vaccine efficacy, which can be used to make a preliminary assessment of vaccine immunization efficacy.
  • a person skilled in the art can select a suitable test method according to factors such as specific test conditions and test purposes.
  • the present application relates to the use of a foot-and-mouth disease vaccine according to the present application for the preparation of a medicament for preventing foot-and-mouth disease in an animal.
  • the animal foot-and-mouth disease disease is foot-and-mouth disease of pig, cow or sheep.
  • the foot and mouth disease of the animal is foot and mouth disease of pigs.
  • the animal foot-and-mouth disease disease is the A serotype of foot and mouth disease of pigs.
  • the present application is directed to a method of preventing foot-and-mouth disease in an animal comprising administering to the animal an immunologically effective amount of a foot-and-mouth disease vaccine according to the present application.
  • immunologically effective amount refers to an amount that elicits an immune response against foot-and-mouth disease virus in an animal to be administered.
  • the immunologically effective amount is related to the species, variety, age, weight and health status of the animal to be administered.
  • the animal foot and mouth disease For foot and mouth disease of pigs, cattle or sheep.
  • the foot and mouth disease of the animal is foot and mouth disease of pigs.
  • the animal foot-and-mouth disease disease is the A serotype of foot and mouth disease of pigs.
  • the present application relates to a foot-and-mouth disease vaccine according to the present application for use in preventing foot-and-mouth disease in an animal.
  • the animal foot-and-mouth disease disease is foot-and-mouth disease of pig, cow or sheep.
  • the foot and mouth disease of the animal is foot and mouth disease of pigs.
  • the animal foot-and-mouth disease disease is the A serotype of foot and mouth disease of pigs.
  • the present application relates to an apparatus for preparing a foot-and-mouth disease vaccine comprising a dual membrane integrated filtration system comprising a microfiltration device and an ultrafiltration device disposed in parallel,
  • the microfiltration device comprises a microfiltration solution tank, a first main pump and a microfiltration membrane, the ultrafiltration device comprising a concentration tank, a second main pump and an ultrafiltration membrane; wherein the microfiltration device is for removing the foot and mouth disease a large particle impurity in the virus cell culture solution, the ultrafiltration device is for removing small molecular impurities in the foot-and-mouth disease virus cell culture solution; the microfiltration device and the ultrafiltration device simultaneously operate on the foot-and-mouth disease virus cell
  • the culture solution is simultaneously subjected to microfiltration and ultrafiltration to form a concentrate containing the foot-and-mouth disease virus in the concentration tank.
  • the microfiltration device and the ultrafiltration device are interconnected such that both the microfiltration filtrate and the ultrafiltration retentate are placed in the concentration tank to form the concentrate containing the
  • the dual membrane integrated filtration system further includes a first constant flow pump and a second constant flow pump, the microfiltration device being coupled to the first constant flow pump, the ultrafiltration device Connected to the second constant current pump to dynamically control the membrane flux.
  • the first constant flow pump is disposed at a filtration end of the microfiltration device
  • the second constant current pump is disposed at a filtration end of the ultrafiltration device.
  • the present embodiment provides a method for preparing a foot-and-mouth disease vaccine, which uses a double membrane combined filtration system to purify the foot-and-mouth disease virus cell culture solution.
  • the specific steps are as follows:
  • the filtration end valve of the microfiltration device and the first constant current pump are turned off, and 259 mL (4.4 times concentrated) is added to the concentration tank at the flow rate of the ultrafiltration filtrate (74.25 mL/min).
  • the antigen concentration and total protein concentration of foot-and-mouth disease virus were detected.
  • the antigenic concentration of foot-and-mouth disease virus was 34.2 ⁇ g/mL, and the removal rate of heteroprotein was over 98%.
  • the total yield of foot-and-mouth disease virus antigen was 98%.
  • the present embodiment provides a method for preparing a foot-and-mouth disease vaccine, which is purified by a double membrane combined filtration system, and further purified (ie, refined) by chromatography, and the specific steps are as follows:
  • the concentrated solution containing the foot-and-mouth disease virus obtained in the step (4) concentration tank is applied to a Capto butyl chromatography packing which has been washed with a PBS buffer solution of pH 7.6 and a conductivity of 90 mS/cm, and continues to be used after loading.
  • the pH 7.6 and the 90 mS/cm PBS buffer solution were rinsed for 5 column volumes, and then the foot-and-mouth disease virus antigen was eluted with a pH 7.6, conductivity 5 mS/cm PBS buffer solution.
  • the concentration of the foot-and-mouth disease virus antigen, the total protein concentration and the nucleic acid residue of the eluted peak were detected.
  • the antigenic concentration of the foot-and-mouth disease virus was 32 ⁇ g/mL, the removal rate of the host nucleic acid was over 99%, and the removal rate of the heteroprotein was over 99%.
  • the total yield was 92%.
  • the present embodiment provides a method for preparing a foot-and-mouth disease vaccine, and adopts a double membrane combined filtration system to carry out large-scale purification of the foot-and-mouth disease virus cell culture solution, and the specific steps are as follows:
  • the amount is controlled at 41.8LMH, and the membrane flux of the ultrafiltration membrane is controlled at 39.6LMH (the difference in membrane flux between the microfiltration membrane and the ultrafiltration membrane is to maintain the same filtrate flow rate), the microfiltration tank And maintaining 200 L of pH 7.6 PBS buffer solution in each of the concentration tanks;
  • the antigen concentration and total protein concentration of foot-and-mouth disease virus were detected.
  • the antigenic concentration of foot-and-mouth disease virus was 30.2 ⁇ g/mL, and the removal rate of heteroprotein was over 94%.
  • the total yield of foot-and-mouth disease virus antigen was 96%.
  • an inactivated and purified foot-and-mouth disease vaccine and an inactivated and purified foot-and-mouth disease vaccine were prepared, and the prepared vaccine was used to carry out an animal immunity test respectively.
  • the specific steps are as follows:
  • Example 1 The purified foot-and-mouth disease virus antigen prepared in Example 1 and the purified foot-and-mouth disease virus pathogen prepared in Example 2 were respectively inactivated with diethyleneimine (BEI), and then blocked with sodium thiosulfate;
  • BEI diethyleneimine
  • Inactivated purified foot-and-mouth disease virus antigen and inactivated purified foot-and-mouth disease virus antigen were respectively sterilized by microfiltration membrane having a pore diameter of 0.22 ⁇ m, and added to pH 7.4 in a volume ratio of 10:7 (antigen solution: buffer solution).
  • the PBST buffer solution ie, PBS buffer solution added with Tween-20
  • the PBST buffer solution was diluted to a concentration of 20 ⁇ g/mL of foot-and-mouth disease virus, and then emulsified by 1:1 weight ratio plus 206 adjuvant, and the inactivated pig foot-and-mouth disease type A was added.
  • Purified vaccine and pig foot-and-mouth disease type A Inactivated refined vaccine;
  • SPF pigs Twelve male non-specific pathogenic pigs (SPF pigs) were selected and divided into two groups, 10 in the experimental group and 2 in the control group.
  • 5 SPF pigs in the experimental group 1 mL of the foot-and-mouth disease type A inactivated purified vaccine was injected into each neck muscle; for the other 5 SPF pigs in the experimental group, the foot-and-mouth disease type A inactivated purified vaccine was injected into each head and neck. 1 mL; 2 SPF pigs in the control group were not immunized.
  • each SPF pig was challenged with 0.5 mL of a type A foot-and-mouth disease virus containing 10 5 TCID50, and the symptoms of foot-and-mouth disease were observed for 15 days.
  • This embodiment provides a method for cleaning and regenerating a microfiltration membrane and an ultrafiltration membrane, and the specific steps are as follows:
  • the pure water is separately added to the microfiltration device and the ultrafiltration device in the double membrane combined filtration system after the use of Examples 1-3 to separately clean the microfiltration membrane and the ultrafiltration membrane, and the membrane is cleaned during the cleaning process.
  • the shear rate is increased to 16000 s -1 , and the membrane flux is gradually increased from 10 LMH to 50 LMH;
  • step (2) After washing in step (1), the 0.1-0.5M NaOH solution is separately added to the microfiltration device and the ultrafiltration device for further cleaning.
  • the solution temperature is 50 ° C
  • the shear rate is 16000 s -1
  • the membrane flux is 50 LMH. Gradually increase to 400LMH for 60 minutes;
  • step (3) After washing in step (2), the microfiltration device and the ultrafiltration device are washed with pure water in a washing mode until the pH of the microfiltration filtrate and the ultrafiltration filtrate is reduced to 9 or less;
  • both the microfiltration membrane and the ultrafiltration membrane maintain good recovery and consistency during 200 uses. Since both microfiltration and ultrafiltration are carried out within the reversible contaminated area of the membrane, the degree of membrane fouling is reduced, the burden of cleaning recovery is reduced, and the stepwise discharge of pollutants and hot water washing prior to the use of the cleaning agent increases the membrane properties. Can reply. Through the measurement of membrane water flux, the use of the membrane within 200 times is in a stable state, which is 5-10 times longer than the membrane life of the conventional process, which will greatly reduce the equipment consumption and reduce the production cost.
  • the invention adopts the double membrane combined integrated filtration system to realize linear amplification under optimized control conditions, and maintains high recovery rate and high impurity removal performance of the small trial integrated process.
  • the volume of the two liquid tanks (ie, the microfiltration liquid tank and the concentration tank) in the dual membrane combined filtration system is close to the volume of the liquid to be concentrated, which is two in comparison with the conventional filtration system.
  • the treatment liquid volume is 10-20 times smaller than the equal volume of the liquid tank, and the time is shortened by half.
  • the vaccine preparation method using the double membrane combined filtration system has the advantages of multi-step operation in the traditional foot-and-mouth disease vaccine preparation process on the basis of maintaining high recovery rate and high purity.

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Abstract

本申请公开了一种口蹄疫疫苗的制备方法，包括如下步骤：(i)获取口蹄疫病毒细胞培养液；(ii)将所述口蹄疫病毒细胞培养液通过双膜联用一体过滤系统进行分离纯化；(iii)收集步骤(ii)中所获得的含有口蹄疫病毒的浓缩液。本申请还公开了由所述方法制备得到的口蹄疫疫苗及其在制备用于预防动物口蹄疫疾病的药物中的用途。本申请还公开了一种用于制备口蹄疫疫苗的装置，其包括双膜联用一体过滤系统。

Description

一种口蹄疫疫苗的制备方法
相关申请的交叉引用
本申请要求申请日为2016年12月7日、申请号为201611113532.1、发明名称为“一种口蹄疫疫苗的制备方法”的中国发明专利申请的优先权，其通过引用整体并入本申请。
技术领域
本申请涉及兽用生物制品领域，具体地，本申请涉及一种口蹄疫疫苗的制备方法、由所述方法制备得到的口蹄疫疫苗、所述口蹄疫疫苗在制备用于预防动物口蹄疫疾病的药物中的用途，以及用于制备口蹄疫疫苗的装置。
背景技术
口蹄疫(Foot and Mouth Disease,FMD)是由口蹄疫病毒引起的一种急性、发热性、高度接触性传染的动物传染病。口蹄疫主要侵害偶蹄类动物，其临床诊断特征为口腔黏膜、蹄部和乳房皮肤发生水疱。目前已开发出多种用于控制和预防口蹄疫疾病的口蹄疫疫苗。对于口蹄疫疫苗的制备方法，目前本领域通常采用的是各种分离纯化加工单元的简单组合，例如，采用连续流离心搭配直流过滤、深层过滤、或者中空纤维过滤、PEG沉淀法或者色谱层析法等手段对收获的口蹄疫病毒细胞培养液进行分离和纯化，以获得纯化的口蹄疫病毒疫苗。
然而，这种分离纯化加工单元的简单组合难以发挥各加工单元的最佳分离纯化效率，造成产品品质、生产效率和生产成本极不平衡的状况，而这是人们所不希望的。
发明概述
本申请涉及一种口蹄疫疫苗的制备方法、由所述方法制备得到的口蹄疫疫苗、所述口蹄疫疫苗在制备用于预防动物口蹄疫疾病的药物中的用途，以及用于制备口蹄疫疫苗的装置。
一方面，本申请提供了一种口蹄疫疫苗的制备方法，包括如下步骤：(i)获取口蹄疫病毒细胞培养液；(ii)将所述口蹄疫病毒细胞培养液通过双膜联用一体过滤系统 进行分离纯化，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液；(iii)收集步骤(ii)中所获得的含有口蹄疫病毒的浓缩液。
在某些实施方式中，所述浓缩罐的体积等于所述口蹄疫病毒细胞培养液的目标浓缩体积或者不大于所述目标浓缩体积的1.5倍。在某些实施方式中，所述浓缩罐的体积为所述口蹄疫病毒细胞培养液初始体积的1/5～1/50。
在某些实施方式中，所述微滤膜的孔径为0.1μm～0.45μm。在某些实施方式中，所述超滤膜是孔径为100kD～500kD的中空纤维超滤膜。
在某些实施方式中，在所述口蹄疫病毒细胞培养液至少部分通过所述微滤装置之后，进一步向所述微滤料液罐中加入第一洗滤液，使其通过所述双膜联用一体过滤系统，获得浓缩液。在某些实施方式中，所述第一洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。在某些实施方式中，所加入的第一洗滤液的体积为微滤截留液体积的1～5倍。在某些实施方式中，将步骤(ii)中所获得的超滤滤过液做为第一洗滤液加入至所述微滤料液罐中，使所述超滤滤过液通过所述双膜联用一体过滤系统，获得浓缩液。
在某些实施方式中，向步骤(ii)中所获得的含有口蹄疫病毒的浓缩液中加入第二洗滤液，使所述浓缩液中的小分子杂质透过超滤膜，获得纯化的病毒浓缩液。在某些实施方式中，所述第二洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。在某些实施方式中，所加入的第二洗滤液的体积为所述病毒浓缩液体积的1～10倍。
在某些实施方式中，所述微滤装置与第一恒流泵连接、所述超滤装置与第二恒流泵连接以动态控制过膜通量。在某些实施方式中，所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端。在某些实施方式中，所述过膜通量为微滤膜或超滤膜的临界膜通量的65％～100％。在某些实施方式中，所述过膜通量为10-150LMH。
在某些实施方式中，所述微滤膜和所述超滤膜的剪切速率各为1500～4000s-1。在 某些实施方式中，所述微滤膜和所述超滤膜的物料与膜面积比均各为10～500L/m2。
在某些实施方式中，在步骤(iii)之后对所述含有口蹄疫病毒的浓缩液进行灭活。在某些实施方式中，在所述灭活之后对所述含有口蹄疫病毒的浓缩液进行乳化。在某些实施方式中，在所述灭活之前或之后对所述含有口蹄疫病毒的浓缩液进行进一步纯化。在某些实施方式中，所述进一步纯化通过PEG沉淀法或色谱层析法进行。在某些实施方式中，所述色谱层析法包括排阻色谱层析法、离子交换色谱层析法、疏水色谱层析法和亲和色谱层析法。
在某些实施方式中，在步骤(iii)之后对所述微滤装置和所述超滤装置进行清洗再生。在某些实施方式中，所述清洗再生包括如下步骤：(a)将纯水分别加入所述微滤装置和所述超滤装置以分别清洗其中的微滤膜和超滤膜，清洗过程中膜剪切速率提升至8000s-1～16000s-1，过膜通量控制在10～500LMH；(b)经步骤(a)清洗后将0.1～0.5M NaOH溶液分别加入所述微滤装置和所述超滤装置做进一步清洗，溶液温度为45～55℃，膜剪切速率为8000s-1～16000s-1，过膜通量控制在50～500LMH，时间为30～60分钟；(c)经步骤(b)清洗后用纯水分别冲洗所述微滤装置和所述超滤装置至微滤滤过液和超滤滤过液的pH值降至9以下。
在某些实施方式中，所述口蹄疫病毒毒株包括一种或多种口蹄疫病毒血清型毒株。在某些实施方式中，所述血清型为O型、A型、C型、SAT1型、SAT2型、SAT3型或Asia1型。
在另一方面，本申请涉及根据本申请所述的方法制备得到的口蹄疫疫苗。
在另一方面，本申请涉及根据本申请所述的口蹄疫疫苗在制备用于预防动物口蹄疫疾病的药物中的用途。
在另一方面，本申请涉及一种预防动物口蹄疫疾病的方法，包括给予所述动物免疫有效量的根据本申请所述的口蹄疫疫苗。
在另一方面，本申请涉及用于预防动物口蹄疫疾病的根据本申请所述的口蹄疫疫苗。
在另一方面，本申请涉及一种用于制备口蹄疫疫苗的装置，其包括双膜联用一体过滤系统，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和 超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液。
在某些实施方式中，所述微滤装置和所述超滤装置相互连接，以使得微滤滤过液和超滤截留液均置于所述浓缩罐中形成所述含有口蹄疫病毒的浓缩液。
在某些实施方式中，所述双膜联用一体过滤系统还包括第一恒流泵和第二恒流泵，所述微滤装置与所述第一恒流泵连接，所述超滤装置与所述第二恒流泵连接以动态控制过膜通量。在某些实施方式中，所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端。
附图说明
图1显示了本申请实施例1的口蹄疫疫苗制备工艺的示意图。
图2显示了本申请实施例2的口蹄疫疫苗制备工艺的示意图。
图3显示了本申请实施例1的口蹄疫病毒抗原SDS-PAGE电泳图。
图4显示了本申请实施例1的口蹄疫病毒抗原HPLC检测结果。
图5显示了本申请实施例5的微滤膜和超滤膜连续使用200次后清洗回复的水通量测定值。
发明详述
尽管本申请将在以下公开多个方面和实施方式，但是在不违背本申请主题精神和范围的前提下，本领域技术人员显然可以对其进行各种等同改变和修改。本申请公开的多个方面和实施方式仅用于举例说明，其并非旨在限制本申请，本申请的实际保护范围以权利要求为准。除非另外指出，本申请中使用的所有技术和科学术语均具有与本申请所属领域中的普通技术人员通常所理解的相同的含义。本申请中引用的所有参考文献、专利、专利申请均通过整体引用并入本文。
一方面，本申请提供了一种口蹄疫疫苗的制备方法，包括如下步骤：(i)获取口蹄疫病毒细胞培养液；(ii)将所述口蹄疫病毒细胞培养液通过双膜联用一体过滤系统进行分离纯化，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所 述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液；(iii)收集步骤(ii)中所获得的含有口蹄疫病毒的浓缩液。
本申请中使用的术语“疫苗”是指含有一种或多种具有机体免疫能力的抗原的组合物。将疫苗引入到宿主动物体内后，该疫苗能激发宿主动物产生针对该一种或多种抗原的免疫反应。
本申请中使用的术语“宿主动物”是指能够被口蹄疫病毒侵入并允许口蹄疫病毒在其体内复制的动物。在某些实施方式中，宿主动物是偶蹄类动物。在某些实施方式中，宿主动物是猪、牛、羊等家畜动物。在某些实施方式中，宿主动物是猪。口蹄疫病毒在宿主动物中的侵入和复制可能使宿主动物产生口蹄疫的临床症状，也可能不使宿主动物产生任何临床症状。
本申请所述的口蹄疫病毒可以包括一种或多种口蹄疫病毒血清型毒株。在某些实施方式中，所述血清型为O型、A型、C型、SAT1型、SAT2型、SAT3型或Asia1型。每种主型又分若干亚型，目前发现的亚型已有70多种。在某些实施方式中，所述血清型为A型。不希望受到任何理论的束缚，本申请的口蹄疫疫苗的制备方法适用于各种口蹄疫病毒血清型毒株或其混合物。
本申请所述的口蹄疫病毒可以是从自然环境中分离纯化获得的天然口蹄疫病毒，也可以是通过基因工程方法获得的重组口蹄疫病毒株，还可以是其他表达系统(工程菌、昆虫、植物)制备的类病毒颗粒、重组抗原。本领域技术人员可以通过本领域公知的基因工程方法获得重组口蹄疫病毒株或者口蹄疫病毒片段，参见，例如，http://www.science.gov/topicpages/v/virus+vaccine+development.html中所描述的。
术语“口蹄疫病毒细胞培养液”是指被口蹄疫病毒感染并允许口蹄疫病毒生长复制的细胞培养液。可以使用本领域公知的方法制备口蹄疫病毒细胞培养液(参见，例如，厍大亮等，口蹄疫灭活疫苗的生产工艺概述，中国兽药杂志，2011 45(1):41～44)。适于培养口蹄疫病毒的细胞包括乳仓鼠肾细胞(BHK21细胞)、猪肾细胞(IBRS-2细胞)、牛肾细胞(MDBK细胞)、非洲绿猴肾细胞(Vero细胞)等。在优选的实施 方式中，本申请使用BHK21细胞培养口蹄疫病毒。
本申请中使用的术语“双膜联用一体过滤系统”是指包括并行设置的微滤装置和超滤装置，并且所述微滤装置和所述超滤装置相互连接、相互配合的一体化过滤系统，其中所述微滤装置包括微滤料液罐、第一主泵和微滤膜；所述超滤装置包括浓缩罐、第二主泵和超滤膜，所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液。在所述双膜联用一体过滤系统中，所述微滤装置和所述超滤装置并排安放并相互连接，以使得微滤滤过液和超滤截留液均置于所述浓缩罐中形成所述含有口蹄疫病毒的浓缩液。
本申请中使用的术语“微滤滤过液”是指所述口蹄疫病毒细胞培养液经过微滤装置之后得到的滤过液。微滤装置是为了除去所述口蹄疫病毒细胞培养液中的大颗粒杂质。当所述口蹄疫病毒细胞培养液经过所述微滤装置之后，所得的微滤滤过液包含的大颗粒杂质水平与在使所述口蹄疫病毒细胞培养液通过所述微滤装置之前的所述口蹄疫病毒细胞培养液中的大颗粒杂质水平相比有显著降低。例如，与在使所述口蹄疫病毒细胞培养液通过所述微滤装置之前的所述口蹄疫病毒细胞培养液中的大颗粒杂质水平相比较，所述微滤装置能除去所述口蹄疫病毒细胞培养液中的至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或者甚至100％的大颗粒杂质。在某些实施方式中，与在使所述口蹄疫病毒细胞培养液通过所述微滤装置之前的所述口蹄疫病毒细胞培养液中的大颗粒杂质水平相比较，所述微滤装置能除去所述口蹄疫病毒细胞培养液中的约30％至约100％、约30％至约90％、约30％至约80％、约40％至约80％、约40％至约90％、约40％至约100％、约50％至约80％、约50％至约90％、约50％至约100％(例如，约60％至约90％、约70％至约90％)、约60％至约100％、约70％至约100％、约80％至约100％、约90％至约100％或约95％至约100％(例如，约96％至约100％、约97％至约100％、约98％至约100％、约99％至约100％、约95％至约96％、约95％至约97％、约95％至约98％或约95％至约99％)的大颗粒杂质。
本申请中使用的术语“大颗粒杂质”是指存在于口蹄疫病毒细胞培养液中的细胞碎片、细菌、聚集体、凝絮体等杂质。在某些实施例中，所述大颗粒杂质的最小直径 大于0.1μm。这些大颗粒杂质的尺寸大于微滤膜的孔径，因此不能通过微滤膜而被截留在微滤料液罐中。
当所述口蹄疫病毒细胞培养液经过微滤装置时，小分子杂质和口蹄疫病毒一起透过微滤膜，存在于微滤滤过液中。微滤滤过液进一步经过超滤装置，超滤装置可以除去所述口蹄疫病毒细胞培养液中的小分子杂质，但是口蹄疫病毒无法通过超滤膜，被截留在超滤截留液中。
本申请中使用的术语“超滤截留液”是指所述微滤滤过液中未通过超滤膜而在超滤装置的浓缩罐中被截留下来的液体，是含有口蹄疫病毒的浓缩液。微滤滤过液经过所述超滤装置之后，所得的超滤截留液包含的小分子杂质水平与在使所述微滤滤过液通过所述超滤装置之前的所述微滤滤过液中的小分子杂质水平相比有显著降低。例如，与在使所述微滤滤过液通过所述超滤装置之前的所述微滤滤过液中的小分子杂质水平相比较，所述超滤装置能除去所述微滤滤过液中的至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、或者甚至100％的小分子杂质。在某些实施方式中，与在使所述微滤滤过液通过所述超滤装置之前的所述微滤滤过液中的小分子杂质水平相比较，所述超滤装置能除去所述微滤滤过液中的约30％至约100％、约30％至约90％、约30％至约80％、约40％至约80％、约40％至约90％、约40％至约100％、约50％至约80％、约50％至约90％、约50％至约100％(例如，约60％至约90％、约70％至约90％)、约60％至约100％、约70％至约100％、约80％至约100％、约90％至约100％或约95％至约100％(例如，约96％至约100％、约97％至约100％、约98％至约100％、约99％至约100％、约95％至约96％、约95％至约97％、约95％至约98％或约95％至约99％)的小分子杂质。
本申请中使用的术语“小分子杂质”是指口蹄疫病毒细胞培养液中的核酸及核酸碎片、宿主蛋白、病毒非结构蛋白、蛋白及病毒降解物、培养基成分等杂质。在某些实施例中，所述小分子杂质的尺寸小于500,000道尔顿。这些小分子杂质的尺寸小于超滤膜的孔径，因此可以透过超滤膜而存在于超滤滤过液中。本申请中使用的术语“超滤滤过液”是指所述微滤滤过液经过超滤装置之后得到的滤过液。
本申请中所述的微滤料液罐用于盛放待分离纯化的口蹄疫病毒细胞培养液，未通过微滤膜的微滤截留液回流至所述微滤料液罐中。本申请中使用的术语“微滤截留 液”是指未通过微滤膜而被截留在微滤料液罐中的液体。在某些实施方式中，所述微滤截留液和待分离纯化的口蹄疫病毒细胞培养液混合置于所述微滤料液罐中。在某些实施方式中，本申请的微滤装置带有搅拌装置，所述搅拌装置进行搅拌将所述微滤料液罐中的微滤截留液和待分离纯化的口蹄疫病毒细胞培养液混合均匀。
本申请所述的浓缩罐用于盛放微滤滤过液，未通过超滤膜的超滤截留液也盛放于浓缩罐中。在某些实施方式中，所述微滤滤过液和超滤截留液混合置于所述浓缩罐中。在某些实施方式中，本申请的超滤装置带有搅拌装置，所述搅拌装置将所述浓缩罐中的微滤滤过液和超滤截留液搅拌混合均匀。
本申请中所述的第一主泵用于将微滤料液罐内的液体不断泵入微滤装置，本申请中所述的第二主泵用于将浓缩罐内的液体不断泵入超滤装置。所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤。在所述双膜联用一体过滤系统中，微滤和超滤同时进行，即在同一时刻，细胞培养液既有一部分在通过微滤膜进行过滤，也有一部分在通过超滤膜进行过滤，而不是像传统制备工艺那样，待所有口蹄疫病毒细胞液均通过微滤装置之后收集微滤滤过液，然后再将微滤滤过液经过超滤装置进行超滤。本申请中的双膜联用一体过滤系统不仅减少了管路衔接，减少了占地面积，而且还大大节省了过滤时间。
在某些实施方式中，进行微滤和超滤的温度均各为2～20℃，例如2℃、3℃、4℃、5℃、6℃、7℃、8℃、9℃、10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃、20℃或者以上任意两个数值之间范围内的任意数值。在优选的实施方式中，进行微滤和超滤的温度均各为2～8℃。恒定的低温控制有助于维持口蹄疫病毒抗原结构的稳定性。
在某些实施方式中，所述浓缩罐的体积等于所述口蹄疫病毒细胞培养液的目标浓缩体积或者不大于所述目标浓缩体积的1.5倍。例如，所述浓缩罐的体积是所述目标浓缩体积的1倍、1.1倍、1.2倍、1.3倍、1.4倍、1.5倍或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所述浓缩罐的体积为所述口蹄疫病毒细胞培养液初始体积的1/5～1/50。例如，所述浓缩罐的体积为所述口蹄疫病毒细胞培养液初始体积的1/5、1/10、1/15、1/20、1/25、1/30、1/35、1/40、1/45、1/50或者以上任意两个数值之间范围内的任意数值。
本申请中所述的微滤膜可以是市售的微滤膜，例如GE Healthcare Life Sciences公司、仕比纯公司、旭化成公司等公司生产的微滤中空纤维膜系列。在某些实施方式中，所述微滤膜的孔径为0.1μm～0.45μm，例如0.1μm、0.15μm、0.2μm、0.22μm、0.25μm、0.3μm、0.35μm、0.4μm、0.45μm或者以上任意两个数值范围之间的任意数值。根据口蹄疫病毒的尺寸，选择尽可能小的微滤膜孔径有助于口蹄疫病毒抗原的回收和大颗粒杂质的去除。
本申请中所述的超滤膜可以是市售的超滤膜，例如GE Healthcare Life Sciences公司、仕比纯公司、旭化成公司等公司生产的超滤中空纤维膜系列。在某些实施方式中，所述超滤膜是孔径为100kD～500kD的中空纤维超滤膜，例如孔径为100kD、150kD、200kD、250kD、300kD、350kD、400kD、450kD、500kD或者以上任意两个数值范围之间任意数值的中空纤维超滤膜。根据口蹄疫病毒的尺寸，选择尽可能大的超滤膜孔径有助于口蹄疫病毒抗原的回收和小分子杂质的去除。
在某些实施方式中，在所述口蹄疫病毒细胞培养液至少部分通过所述微滤装置之后，进一步向所述微滤料液罐中加入第一洗滤液，使其通过所述双膜联用一体过滤系统，获得浓缩液。其中所述“至少部分”是指至少30％、至少40％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％、甚至100％或者以上任意两个数值范围之间的任意数值。加入第一洗滤液有利于残留在微滤料液罐中的口蹄疫病毒抗原通过微滤膜进入超滤装置进行富集，通过第一洗滤液的洗滤可以增加口蹄疫病毒抗原在微滤过程中的回收率。
在某些实施方式中，所述第一洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。在某些实施方式中，所述缓冲溶液为磷酸盐缓冲溶液(PBS)、硼酸盐缓冲溶液、柠檬酸盐缓冲溶液、醋酸盐缓冲溶液、巴比妥酸盐缓冲溶液或者三羟甲基氨基甲烷缓冲液(Tris)。在优选的实施方式中，所述缓冲溶液是PBS缓冲溶液。在某些实施方式中，所述缓冲溶液的pH为7.2、7.5、8.0、8.5、9.0或者以上任意两个数值之间范围内的任意数值。在某些实施方式中，所述缓冲溶液的电导为5mS/cm、50mS/cm、100mS/cm、150mS/cm、200mS/cm、250mS/cm、300mS/cm或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所加入的第一洗滤液的体积为微滤截留液体积的1～5倍，例 如1倍、2倍、3倍、4倍、5倍或者以上任意两个数值之间范围内的任意数值。在某些实施方式中，使用的第一洗滤液的量可以是细胞培养液的初始体积的1/5倍、1/2倍、1倍或者2倍。
在某些实施方式中，将经过微滤和超滤后所获得的超滤滤过液做为第一洗滤液加入至所述微滤料液罐中，洗滤微滤膜截留的口蹄疫病毒抗原进入超滤装置，获得浓缩液。这样不仅可以合理回收利用资源，大幅降低洗滤液的用量，进一步降低成本，还可增加口蹄疫病毒抗原的回收率。
在某些实施方式中，向经过微滤和超滤后所获得的含有口蹄疫病毒的浓缩液中加入第二洗滤液，进一步使所述浓缩液中残留的小分子杂质通过超滤膜，获得纯化的病毒浓缩液。加入所述第二洗滤液有利于将超滤装置中的小分子杂质冲洗出来，减少小分子杂质在超滤截留液中的残留，从而提高口蹄疫病毒浓缩液中口蹄疫病毒的纯度。
在某些实施方式中，所述第二洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。在某些实施方式中，所述缓冲溶液为磷酸盐缓冲溶液(PBS)、硼酸盐缓冲溶液、柠檬酸盐缓冲溶液、醋酸盐缓冲溶液、巴比妥酸盐缓冲溶液或者三羟甲基氨基甲烷缓冲液(Tris)。在优选的实施方式中，所述缓冲溶液是磷酸盐缓冲溶液。在某些实施方式中，所述缓冲溶液的pH为7.2、7.5、8.0、8.5、9.0或者以上任意两个数值之间范围内的任意数值。在某些实施方式中，所述缓冲溶液的电导为5mS/cm、50mS/cm、100mS/cm、150mS/cm、200mS/cm、250mS/cm、300mS/cm或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所加入的第二洗滤液的体积为所述口蹄疫病毒浓缩液体积的1～10倍，例如1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或者以上任意两个数值之间范围内的任意数值。本申请中使用的术语“口蹄疫病毒浓缩液”或者“含有口蹄疫病毒的浓缩液”是指经过微滤和超滤处理之后而降低了大颗粒杂质以及小分子杂质的含量的、未通过超滤膜而被截留在浓缩罐中的液体。
在某些实施方式中，所述微滤装置与第一恒流泵连接、所述超滤装置与第二恒流泵连接以动态控制过膜通量。本申请中使用的术语“动态控制”是指通过动态地改变过膜通量来维持微滤膜和超滤膜表面污染处于不可逆污染阈值及其以下，比如随着浓缩倍数的提高，需要不断调节恒流泵以降低过膜通量从而使膜表面污染处于不可逆污染状态，从而延缓膜的效率下降，延长膜的使用寿命。本申请中使用的术语“过膜通 量”是指单位时间内通过单位膜面积上的流体量。在本申请中，过膜通量的单位用LMH(L/m^2*h)表示，即在一小时之内通过一平方米的膜面积的流体体积数。
本申请中所述的第一恒流泵用于控制微滤装置的过膜通量，所述的第二恒流泵用于控制超滤装置的过膜通量。在某些实施方式中，所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端，从而实现对过膜通量的动态控制。
可以通过控制口蹄疫病毒细胞培养液的浓缩倍数(即细胞培养液浓缩后的体积除以细胞培养液的初始体积所得之倍数)来控制过膜通量。优选地，当浓缩倍数为1/5～1/50时，过膜通量可以维持一个可以操作的合理范围，否则浓缩倍数过高会导致过膜通量过低，操作时间过长；而过低的浓缩倍数会使口蹄疫病毒抗原回收低，需要使用大量洗滤液进行洗滤。
在某些实施方式中，所述过膜通量为微滤膜或超滤膜的临界膜通量以下。本申请中使用的术语“临界膜通量”是微滤膜或超滤膜处于可逆污染和不可逆污染的临界值。当过膜通量在临界膜通量之下时，微滤膜或超滤膜处于可逆污染状态；而当过膜通量在临界膜通量之上时，微滤膜或超滤膜将处于不可逆污染状态。临界膜通量由于膜的孔径、材质、结构的不同而有所不同，而且样品状态和操作条件也会影响到临界膜通量。可以通过本领域已知的多种方法测定膜的临界膜通量，比如通过梯度增加过膜通量检测记录过膜压力的方式，或者通过梯度增加过膜压力检测记录过膜通量的方式(参见，例如，R.W.Field et al.,Critical flux concept for microfiltration fouling,Journal of Membrane Science,1995,100(3):259-272)。
在分离纯化过程中，过膜通量是影响口蹄疫病毒的产率和膜寿命的重要因素之一。如果过膜通量超过临界膜通量，则可能会导致膜的不可逆污染，从而降低膜的使用寿命；如果过膜通量过于低于临界膜通量，则可能会导致生产时间不断延长，生产体系稳定性维持成本加大，导致生产效率过低。因此，需要在临界膜通量以下选择合适的过膜通量，从而保证口蹄疫病毒的高收率和杂质的去除，也可保证系统控制的稳定性，维持较长的使用寿命，从而大幅降低生产成本。在某些实施方式中，所述过膜通量是微滤膜或超滤膜的临界膜通量的65％～100％，例如，过膜通量是微滤膜或超滤膜的临界膜通量的65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％或者以上任意两个数值之间范围内的任意数值。在某些实施 方式中，所述过膜通量为10-150LMH，例如，10LMH、20LMH、30LMH、40LMH、50LMH、60LMH、70LMH、80LMH、90LMH、100LMH、110LMH、120LMH、130LMH、140LMH、150LMH或者以上任意两个数值之间范围内的任意数值。
流体通过所述微滤膜和所述超滤膜时的剪切速率也是影响口蹄疫病毒的产率和膜寿命的重要因素之一。若采用过大的剪切速率，由于大于口蹄疫病毒抗原剪切耐受范围，会导致病毒的剪切破坏和解离，降低收率；而过小的剪切速率会降低过膜通量，降低膜分离的效率。在某些实施方式中，所述微滤膜和所述超滤膜的剪切速率各为1500～4000s-1，该剪切速率下可使口蹄疫病毒抗原的损失不超过5％。例如所述微滤膜和所述超滤膜的剪切速率各为1500s-1、1600s-1、1700s-1、1800s-1、1900s-1、2000s-1、2100s-1、2200s-1、2300s-1、2400s-1、2500s-1、2600s-1、2700s-1、2800s-1、2900s-1、3000s-1、3100s-1、3200s-1、3300s-1、3400s-1、3500s-1、3600s-1、3700s-1、3800s-1、3900s-1、4000s-1或者以上任意两个数值之间范围内的任意数值。
所述微滤膜和所述超滤膜的物料与膜面积比也是影响产率和膜寿命的重要因素之一。物料与膜面积比的计算方式是待处理料液体积除以膜面积。如果物料与膜面积比过高，则可能超出膜载量，使临界膜通量及其以下的操作条件难以维持，导致膜不可逆污染，或者操作时间过长，生产效率低下；如果物料与膜面积比过低，则膜效率未被充分发挥，增加生产成本。因此，需要选择合适的物料与膜面积比，从而在确保高收率的前提下能尽可能地延长膜的使用寿命。在某些实施方式中，所述微滤膜和所述超滤膜的物料与膜面积比均各为10～500L/m2，例如10L/m2、50L/m2、100L/m2、150L/m2、200L/m2、250L/m2、300L/m2、350L/m2、400L/m2、450L/m2、500L/m2或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，在上述步骤(iii)之后对所述含有口蹄疫病毒的浓缩液进行灭活。可以采用本领域常用的方法对含有口蹄疫病毒的浓缩液进行灭活，例如，将浓缩纯化得到的含有口蹄疫病毒的浓缩液用1mM～3mM的二乙烯亚胺(BEI)30℃灭活28h，之后按1.6％体积比加入阻断剂硫代硫酸钠，阻断20min，4℃备用；或者将1/4000β丙内酯加入浓缩纯化得到的含有口蹄疫病毒的浓缩液在37℃灭活3～4天，存储于4℃备用(参见，例如，厍大亮等，口蹄疫灭活疫苗的生产工艺概述，中国兽药杂志，2011 45(1):41～44)。
在某些实施方式中，在所述灭活之后对所述含有口蹄疫病毒的浓缩液进行乳化。 可以采用本领域常用的方法对含有口蹄疫病毒的浓缩液进行乳化，例如先制备水相，即，使用灭菌处理过的生理盐水稀释所述含有口蹄疫病毒的浓缩液，备用；再制备油相，即，将油相佐剂，例如Montanide ISA 206在120℃下灭菌后备用；然后将水相和油相预热至30℃，按重量比1：1配置，在低速搅拌的情况下，将预热至30℃的水相缓慢加入到同样预热至30℃的油相中，乳化20分钟左右，使水相与油相充分混合，乳化成双向型(W/O/W)油乳剂疫苗，放置于4℃保存。
在某些实施方式中，在所述灭活之前或之后对所述含有口蹄疫病毒的浓缩液进行进一步纯化。在某些实施方式中，所述进一步纯化通过PEG(聚乙二醇)沉淀法或色谱层析法进行。
PEG沉淀法是通过加入PEG改变含有口蹄疫病毒的浓缩液的理化参数，控制浓缩液中各种成分的溶解度，从而将浓缩液中的口蹄疫病毒和其他杂质分开的技术。常用的沉淀剂有PEG2000、PEG4000、PEG6000等。一般操作步骤包括：向含有口蹄疫病毒的浓缩液中加入PEG形成沉淀，让生成的沉淀与母液一起放置一段时间促进沉淀粒子生成，然后再离心或过滤，收集沉淀物。
在某些实施方式中，所述色谱层析法包括排阻色谱层析法、离子交换色谱层析法、疏水色谱层析法和亲和色谱层析法。
排阻色谱层析法又称为凝胶色谱法，是利用含有口蹄疫病毒的浓缩液中口蹄疫病毒和其他杂质的分子量大小的不同和在填料上渗透程度的不同，以使口蹄疫病毒和其他杂质相分离。用于凝胶色谱法的填料包括聚丙烯酰胺凝胶、交联葡聚糖凝胶、琼脂糖凝胶、聚苯稀凝胶、硅胶、聚甲基丙烯酸酯等。各种凝胶色谱填料也均是有市售的，例如GE Healthcare Life Sciences公司、Tosho公司、Merck Millipore公司、博格隆公司、纳微科技公司、西安蓝晓公司等生产的凝胶色谱填料。在某些实施方式中，所采用的凝胶色谱填料是交联葡聚糖凝胶。采用凝胶色谱法对含有口蹄疫病毒的浓缩液进行进一步纯化一般是将凝胶色谱填料加入到含有口蹄疫病毒的浓缩液中，然后再将填料装入色谱柱中进行色谱操作，或者是将含有口蹄疫病毒的浓缩液直接加入到装有凝胶色谱填料的色谱柱中进行色谱操作。
离子交换色谱法是利用离子交换色谱填料上的带电基团与含有口蹄疫病毒的浓缩液中不同带电组分的相互作用，将口蹄疫病毒与其他杂质相分离，从而达到进一步纯化口蹄疫病毒的目的。根据离子交换色谱填料表面的电荷不同可以分为阳离子交换色 谱和阴离子交换色谱。在某些实施方式中，所采用的离子交换色谱填料表面的离子交换基团为二乙基氨基乙基(DEAE)、季铵基(Q)、二乙基氨基丙基(ANX)、羧甲基(CM)、磺酸基(SP)等。各种离子交换色谱填料也均是有市售的，例如GE Healthcare Life Sciences公司、Tosho公司、Merck Millipore公司、博格隆公司、纳微科技公司、西安蓝晓公司等生产的离子交换色谱填料。采用离子交换色谱法对含有口蹄疫病毒的浓缩液进行进一步纯化一般是将离子交换色谱填料加入到含有口蹄疫病毒的浓缩液中，然后再将填料装入色谱柱中进行色谱操作，或者是将含有口蹄疫病毒的浓缩液直接加入到装有离子色谱填料的色谱柱中进行色谱操作。
疏水色谱层析法是利用疏水作用色谱填料上的疏水基团与含有口蹄疫病毒的浓缩液中的口蹄疫病毒表面的疏水基团的相互作用，实现口蹄疫病毒在色谱填料上的吸附，从而与浓缩液中不发生吸附和吸附较弱的杂质相分离。在某些实施方式中，所采用的疏水色谱填料表面的疏水基团为丁基、丁硫基、苯基、或辛基。各种疏水色谱填料也均是有市售的，例如GE Healthcare Life Sciences公司、Tosho公司、Merck Millipore公司、博格隆公司、纳微科技公司、西安蓝晓公司等生产的疏水色谱填料。采用疏水色谱层析法对含有口蹄疫病毒的浓缩液进行进一步纯化一般是将疏水作用色谱填料加入到含有口蹄疫病毒的浓缩液中，然后再将填料装入色谱柱中进行色谱操作，或者是将含有口蹄疫病毒的浓缩液直接加入到装有疏水作用色谱填料的色谱柱中进行色谱操作。
亲和色谱层析法是将具有特殊结构的亲和分子填料制成固相吸附剂放置在层析柱中，当含有口蹄疫病毒的浓缩液通过层析柱时，与吸附剂具有亲和能力的口蹄疫病毒就会被吸附而滞留在层析柱中，那些没有亲和力的杂质由于不被吸附而直接流出，从而实现口蹄疫病毒与杂质的分离。然后再选用适当的洗脱液，改变结合条件将被结合的口蹄疫病毒洗脱下来。在某些实施方式中，使用的吸附剂为氧化铝、硅胶、聚酰胺等。各种亲和分子填料也均是有市售的，例如肝素亲和介质。采用亲和色谱层析法对含有口蹄疫病毒的浓缩液进行进一步纯化一般是将亲和分子填料加入到含有口蹄疫病毒的浓缩液中，然后再将填料装入色谱柱中进行色谱操作，或者是将含有口蹄疫病毒的浓缩液直接加入到装有亲和分子填料的色谱柱中进行色谱操作。
在某些实施方式中，在上述步骤(iii)之后对所述微滤装置和所述超滤装置进行清洗再生。所述微滤装置中的微滤膜和超滤装置中的超滤膜在长期运行过程中会导致膜 的透水量随着运行时间的增加而降低，即产生膜污染。因此，有必要对微滤膜和超滤膜进行清洗再生，回复膜的性能，延长膜的使用寿命，从而降低生产成本。
在某些实施方式中，所述清洗再生包括如下步骤：(a)将纯水分别加入所述微滤装置和所述超滤装置以分别清洗其中的微滤膜和超滤膜，清洗过程中膜剪切速率提升至8000s-1～16000s-1，过膜通量控制在10～500LMH；(b)经步骤(a)清洗后将0.1～0.5M NaOH溶液分别加入所述微滤装置和所述超滤装置做进一步清洗，溶液温度为45～55℃，膜剪切速率为8000s-1～16000s-1，过膜通量控制在50～500LMH，时间为30～60分钟；(c)经步骤(b)清洗后用纯水分别冲洗所述微滤装置和所述超滤装置至微滤滤过液和超滤滤过液的pH值降至9以下。
在某些实施方式中，所述步骤(a)中膜剪切速率提升至8000s-1、9000s-1、10000s-1、11000s-1、12000s-1、13000s-1、14000s-1、15000s-1、16000s-1或者以上任意两个数值之间范围内的任意数值，所述过膜通量控制在10LMH、50LMH、100LMH、150LMH、200LMH、250LMH、300LMH、350LMH、400LMH、450LMH、500LMH或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所述步骤(b)中NaOH溶液温度为45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃或者以上任意两个数值之间范围内的任意数值；膜剪切速率为8000s-1、9000s-1、10000s-1、11000s-1、12000s-1、13000s-1、14000s-1、15000s-1、16000s-1或者以上任意两个数值之间范围内的任意数值；所述过膜通量控制在50LMH、100LMH、150LMH、200LMH、250LMH、300LMH、350LMH、400LMH、450LMH、500LMH或者以上任意两个数值之间范围内的任意数值；所述时间为30分钟、35分钟、40分钟、45分钟、50分钟、55分钟、60分钟或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所述步骤(c)中的pH值降至9、8.5、8.0、7.5、7.0或者以上任意两个数值之间范围内的任意数值。
在某些实施方式中，所述清洗再生过程中仍然对过膜通量进行动态控制以避免污染物在高跨膜压下深入膜孔隙，导致难以清除。而且温热强碱对污染物具有分解作用，从而使得膜性能得到非常好的维护。
在另一方面，本申请涉及根据本申请所述的方法制备得到的口蹄疫疫苗。在某些实施方式中，本申请的口蹄疫疫苗可以通过适当的途径递送到给药动物中，包括但不 限于，通过口服途径、注射途径(如静脉注射、肌肉注射、皮下注射、皮内注射、心内注射、鞘内注射、胸膜腔内注射、腹腔内注射等)、粘膜途径(如鼻腔内给药、口腔内给药等)、舌下途径、直肠途径、经皮途径、眼内途径、肺部途径。在某些实施方式中，本申请的口蹄疫疫苗可通过注射途径给药。如本领域技术人员所知，口蹄疫疫苗的用量依活性成分的活性、给药动物个体的年龄、体重等因素的不同而变化。本领域技术人员可以根据前述影响用量的因素容易地确定最合适的疫苗用量。
根据本申请所述的方法制备得到的口蹄疫疫苗可以通过已知的多种方法来检测其免疫效力。现有技术提供了多种评估疫苗免疫效力的试验方法，例如，攻毒保护试验、血清学试验等。攻毒保护试验直接采用强病毒原对疫苗接种后的试验动物进行人工感染，然后根据动物的发病情况来判断疫苗的免疫效力。攻毒保护试验是检验疫苗免疫效力的最直接的方法，因此包括中国在内的多个国家都要求采用攻毒保护试验来评价兽用疫苗的免疫效力。有关攻毒保护试验的具体操作方法可参见，2010版《中国兽药典》中有关半数保护量(PD50)测定的章节。也可以采用血清学试验方法来评估疫苗的免疫效力，例如通过ELISA法(即酶联免疫吸附测定法)测定免疫动物血清中产生的抗体含量进而判断疫苗的免疫效力。采用ELISA法测定时通常是先将抗原(或抗体)结合在固相载体上，然后加一种抗体(或抗原)与酶结合成的偶联物(标记物)，当偶联物与固相载体上的抗原(或抗体)反应结合后再加上酶的相应底物，即起催化水解或氧化还原反应而呈颜色，其所生成的颜色深浅与待测的抗原(或抗体)含量成正比。这些血清学指标与疫苗效力之间存在关联性，其可以用来对疫苗免疫效力进行初步评估。本领域技术人员可以根据具体的试验条件以及试验目的等因素来选择合适的试验方法。
在另一方面，本申请涉及根据本申请所述的口蹄疫疫苗在制备用于预防动物口蹄疫疾病的药物中的用途。在某些实施方式中，所述动物口蹄疫疾病为猪、牛或羊口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫的A血清型。
在另一方面，本申请涉及一种预防动物口蹄疫疾病的方法，包括给予所述动物免疫有效量的根据本申请所述的口蹄疫疫苗。本申请中使用的术语“免疫有效量”是指在给药动物体内引起针对口蹄疫病毒的免疫反应的量。免疫有效量与给药动物的物种、品种、年龄、体重和健康状况等因素有关。在某些实施方式中，所述动物口蹄疫疾病 为猪、牛或羊口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫的A血清型。
在另一方面，本申请涉及用于预防动物口蹄疫疾病的根据本申请所述的口蹄疫疫苗。在某些实施方式中，所述动物口蹄疫疾病为猪、牛或羊口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫。在某些实施方式中，所述动物口蹄疫疾病为猪口蹄疫的A血清型。
在另一方面，本申请涉及一种用于制备口蹄疫疫苗的装置，其包括双膜联用一体过滤系统，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液。在某些实施方式中，所述微滤装置和所述超滤装置相互连接，以使得微滤滤过液和超滤截留液均置于所述浓缩罐中形成所述含有口蹄疫病毒的浓缩液。
在某些实施方式中，所述双膜联用一体过滤系统还包括第一恒流泵和第二恒流泵，所述微滤装置与所述第一恒流泵连接，所述超滤装置与所述第二恒流泵连接以动态控制过膜通量。在某些实施方式中，所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端。
具体实施方式
下面结合具体实施例对本发明进行详细说明。所有实施例中涉及的生物学材料如口蹄疫病毒毒株、培养液、工具酶、缓冲溶液，以及各种培养方法、病毒灭活、纯化、精制等工艺均是本领域技术人员所熟悉的，可以参考Sambrook等人编著的“分子克隆”(实验室手册，冷泉港，1989)及“精编分子生物学实验指南”(美/F.奥斯伯等著，颜子颖等译，北京，科学出版社，1998)。
实施例1
本实施例提供了一种口蹄疫疫苗的制备方法，采用双膜联用一体过滤系统对口蹄疫病毒细胞培养液进行纯化，具体步骤如下：
(1)按图1搭建双膜联用一体过滤系统，采用pH7.6的PBS缓冲溶液平衡微滤膜(孔径：0.2μm，内径：1mm，膜面积：110cm2)和超滤膜(孔径：500kD，内径：1mm，膜面积：110cm2)，并将微滤膜和超滤膜的剪切速率均控制在4000s-1，将温度控制在2～10℃，同时将微滤膜和超滤膜的过膜通量均控制在63LMH，在浓缩罐中维持58mL pH7.6的PBS缓冲溶液；
(2)按80L/m2的物料/膜面积比，将经过BHK21细胞悬浮培养的口蹄疫病毒A/GDMM/2013(抗原浓度为2.3μg/mL)细胞培养液加入至微滤料液罐中，使其通过双膜联用一体过滤系统进行微滤超滤一体化处理，至口蹄疫病毒细胞培养液体积减少至初始体积的1/5时，将微滤膜和超滤膜的过膜通量同时调至49.5LMH，待口蹄疫病毒细胞培养液体积减少至初始体积的1/10时，将微滤膜和超滤膜的过膜通量同时调至40.5LMH；
(3)待口蹄疫病毒细胞培养液体积减少至初始体积的1/15时，以等超滤滤过液流速(74.25mL/min)向微滤料液罐中加入118mL(2倍浓缩罐体积的洗滤液)pH7.6、电导为31mS/cm的PBS缓冲溶液作为第一洗滤液进行微滤洗滤；
(4)微滤洗滤结束后，关闭微滤装置的滤过端阀门和第一恒流泵，以等超滤滤过液流速(74.25mL/min)向浓缩罐中加入259mL(4.4倍浓缩罐体积的洗滤液)pH7.6、电导为31mS/cm的PBS缓冲溶液作为第二洗滤液进行超滤洗滤；
(5)收集浓缩罐中含有口蹄疫病毒的浓缩液，其中口蹄疫病毒抗原的十二烷基硫酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳图如图3所示，高效液相色谱(HPLC)检测结果如图4所示。
检测口蹄疫病毒抗原浓度及总蛋白浓度，测得口蹄疫病毒抗原浓度为34.2μg/mL，其杂蛋白去除率在98％以上，所得口蹄疫病毒抗原总收率为98％。
实施例2
本实施例提供了一种口蹄疫疫苗的制备方法，采用双膜联用一体过滤系统对口蹄疫病毒细胞培养液进行纯化，并利用色谱层析法进行进一步纯化(即，精制)，具体步骤如下：
(1)按图2搭建双膜联用一体过滤系统，采用pH7.6的PBS缓冲溶液平衡微滤膜(孔径：0.45μm，内径：1mm，膜面积：110cm2)和超滤膜(孔径：300kD，内径： 1mm，膜面积：110cm2)，并将微滤膜和超滤膜的剪切速率均控制在1500s-1，将温度控制在2～10℃，同时将微滤膜和超滤膜的过膜通量控制在36LMH，在浓缩罐中预先加入58mL pH7.6的PBS缓冲溶液；
(2)按80L/m2物料/膜面积比，将经过BHK21细胞悬浮培养的口蹄疫病毒A/GDMM/2013(抗原浓度为2.3μg/mL)细胞培养液加入至微滤料液罐中，使其通过双膜联用一体过滤系统进行微滤超滤一体化处理，至口蹄疫病毒细胞培养液体积减少至初始体积的1/5时，将微滤膜和超滤膜的过膜通量同时调至28.2LMH，待口蹄疫病毒细胞培养液体积减少至初始体积的1/10时，将微滤膜和超滤膜的过膜通量同时调至23.1LMH；
(3)待口蹄疫病毒细胞培养液体积减少至初始体积的1/15时，以等超滤滤过液流速(42.4mL/min)，将118mL(2倍浓缩罐体积的洗滤液)超滤滤过液回流至微滤料液罐作为第一洗滤液进行微滤洗滤；
(4)微滤洗滤结束后，关闭微滤装置的滤过端阀门及第一恒流泵，以等超滤滤过液流速(42.4mL/min)向浓缩罐中加入259mL(4.4倍浓缩罐体积的洗滤液)pH7.6、电导100mS/cm的PBS缓冲溶液作为第二洗滤液进行超滤洗滤；
(5)将步骤(4)浓缩罐中所获得的含有口蹄疫病毒的浓缩液上样至经pH7.6、电导90mS/cm的PBS缓冲溶液淋洗过的Capto butyl色谱填料，上样后继续用pH7.6、电导90mS/cm的PBS缓冲溶液淋洗5个柱体积，然后用pH7.6、电导5mS/cm的PBS缓冲溶液洗脱口蹄疫病毒抗原。
检测洗脱峰的口蹄疫病毒抗原浓度、总蛋白浓度和核酸残留，测得口蹄疫病毒抗原浓度为32μg/mL，宿主核酸去除率在99％以上，杂蛋白去除率在99％以上，所得口蹄疫病毒抗原总收率为92％。
实施例3
本实施例提供了一种口蹄疫疫苗的制备方法，采用双膜联用一体过滤系统对口蹄疫病毒细胞培养液进行规模化纯化，具体步骤如下：
(1)按图1搭建双膜联用一体过滤系统，采用pH7.6的PBS缓冲溶液平衡微滤膜(孔径：0.2μm，内径：1mm，膜面积：38m2)和超滤膜(孔径：500kD，内径：1mm，膜面积：36m2)，并将微滤膜和超滤膜的剪切速率均控制在2000s-1，将温度控制在2～ 10℃，将微滤膜的过膜通量控制在41.8LMH，将超滤膜的过膜通量控制在39.6LMH(微滤膜和超滤膜的过膜通量的差异是为了保持相同的滤过液流速)，微滤料液罐和浓缩罐中各维持200L pH7.6的PBS缓冲溶液；
(2)以23.76L/min的流速向微滤料液罐中加入经过BHK21细胞悬浮培养的口蹄疫病毒A/GDMM/2013(抗原浓度为2.1μg/mL)细胞培养液，使其通过双膜联用一体过滤系统进行微滤超滤一体处理。待泵入2400L口蹄疫病毒细胞培养液进入双膜联用一体过滤系统时，将进料速度调至19.8L/min，并将微滤膜的过膜通量调至31.2LMH，同时将超滤膜的过膜通量调至33LMH。待再次泵入300L口蹄疫病毒细胞培养液进入双膜联用一体过滤系统时，将进料速度调至16.2L/min，将微滤膜的过膜通量调至25.5LMH，同时将超滤膜的过膜通量调至27LMH；
(3)待口蹄疫病毒细胞培养液全部(共3000L)泵入双膜联用一体过滤系统时，以等超滤滤过液流速(16.2L/min)向微滤料液罐中加入400L(2倍浓缩罐体积的洗滤液)pH7.6、电导为31mS/cm的PBS缓冲溶液作为第一洗滤液进行微滤洗滤；
(4)微滤洗滤结束后，关闭微滤装置的滤过端，以等超滤滤过液流速(16.2L/min)向浓缩罐中加入1000L(5倍浓缩罐体积的洗滤液)pH7.6、电导为31mS/cm的PBS缓冲溶液作为第二洗滤液进行超滤洗滤；
(5)收集浓缩罐中含有口蹄疫病毒的浓缩液。
检测口蹄疫病毒抗原浓度及总蛋白浓度，测得口蹄疫病毒抗原浓度为30.2μg/mL，杂蛋白去除率在94％以上，所得口蹄疫病毒抗原总收率为96％。
实施例4
本实施例制备了灭活纯化口蹄疫疫苗和灭活精制口蹄疫疫苗，并用制备的疫苗分别进行动物免疫效力实验，具体步骤如下：
(1)将实施例1制备得到的纯化口蹄疫病毒抗原和实施例2制备得到的精制口蹄疫病毒病原分别用二乙烯亚胺(BEI)灭活后，加入硫代硫酸钠阻断；
(2)灭活的纯化口蹄疫病毒抗原和灭活的精制口蹄疫病毒抗原分别过孔径为0.22μm的微滤膜除菌，按10：7的体积比(抗原溶液：缓冲溶液)各加入pH7.4的PBST缓冲溶液(即，加入吐温-20的PBS缓冲溶液)稀释至口蹄疫病毒抗原浓度为20μg/mL，再按1:1重量比加206佐剂乳化，分装得猪口蹄疫A型灭活纯化疫苗和猪口蹄疫A型 灭活精制疫苗；
(3)选择12头雄性无特定病原猪(SPF猪)，分成两组，实验组10头，对照组2头。对于实验组中的5头SPF猪，每头颈部肌肉注射猪口蹄疫A型灭活纯化疫苗1mL；对于实验组中的另外5头SPF猪，每头颈部注射猪口蹄疫A型灭活精制疫苗1mL；对照组中的2头SPF猪不进行任何免疫。第28天，对每头SPF猪用0.5mL含105TCID50的流行A型口蹄疫病毒攻击，观察猪口蹄疫症状15天。
结果表明，实验组的所有免疫猪均未见不良反应，未见口蹄疫症状出现，而对照组的未免疫猪全部死亡。由此可见，用本申请的方法制备得到的猪口蹄疫A型灭活纯化疫苗和猪口蹄疫A型灭活精制疫苗的免疫保护率均为100％。而且，本实施例还表明，采用本发明的双膜联用一体过滤系统制备得到的口蹄疫病毒抗原已达到动物安全免疫的纯度，可无需进一步纯化即可制备疫苗。
实施例5
本实施例提供了微滤膜和超滤膜的清洗再生方法，具体步骤如下：
(1)将纯水分别加入实施例1-3使用后的双膜联用一体过滤系统中的微滤装置和超滤装置以分别清洗其中的微滤膜和超滤膜，清洗过程中将膜剪切速率提升至16000s-1，将过膜通量由10LMH逐步提升至50LMH；
(2)经步骤(1)清洗后将0.1～0.5M NaOH溶液分别加入微滤装置和超滤装置做进一步清洗，溶液温度为50℃，剪切速率为16000s-1，过膜通量由50LMH逐步提升至400LMH，时间为60分钟；
(3)经步骤(2)清洗后用纯水以洗滤模式冲洗微滤装置和超滤装置至微滤滤过液和超滤滤过液的pH降至9以下；
(4)将剪切流速降低至2000s-1，温度控制在20℃，将过膜通量由5LMH逐步提升至100LMH，测定系统的水通量并记录。
(5)检测记录200次使用后的微滤膜和超滤膜的水通量，如图5所示。
由图5可见，在200次的使用过程中，微滤膜和超滤膜均保持较好的回复性和一致性。由于微滤和超滤均在膜的可逆污染区域以内进行，降低了膜污染程度，减轻了清洗回复的负担，同时在使用清洁剂之前分步排放污染物和热水冲洗均提高了膜的性 能回复。通过膜水通量的测定，膜在200次以内的使用处于稳定状态，比传统工艺的膜寿命长5-10倍，这将大幅降低设备耗材投入，降低生产成本。
综上所述，本发明采用双膜联用一体过滤系统在优化的控制条件下实现了线型放大，并且保持小试一体工艺高回收率，高杂质去除的性能。在规模生产工艺中，双膜联用一体过滤系统中的两个料液罐(即微滤料液罐和浓缩罐)体积接近待浓缩后的料液体积，比传统过滤系统中两个与预处理料液等体积的料液罐相比小了10-20倍，时间缩短一半。最为重要的是，本发明采用双膜联用一体过滤系统的疫苗制备方法在保持高回收率和高纯度的基础上，一步实现了传统口蹄疫疫苗制备工艺中多步操作的效果。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。

Claims (33)

  1. 一种口蹄疫疫苗的制备方法，包括如下步骤：
    (i)获取口蹄疫病毒细胞培养液；
    (ii)将所述口蹄疫病毒细胞培养液通过双膜联用一体过滤系统进行分离纯化，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液；
    (iii)收集步骤(ii)中所获得的含有口蹄疫病毒的浓缩液。
  2. 根据权利要求1所述的方法，其中所述浓缩罐的体积等于所述口蹄疫病毒细胞培养液的目标浓缩体积或者不大于所述目标浓缩体积的1.5倍。
  3. 根据权利要求1所述的方法，其中所述浓缩罐的体积为所述口蹄疫病毒细胞培养液初始体积的1/5～1/50。
  4. 根据前述权利要求中任一项所述的方法，其中所述微滤膜的孔径为0.1μm～0.45μm。
  5. 根据前述权利要求中任一项所述的方法，其中所述超滤膜是孔径为100kD～500kD的中空纤维超滤膜。
  6. 根据前述权利要求中任一项所述的方法，其中在所述口蹄疫病毒细胞培养液至少部分通过所述微滤装置之后，进一步向所述微滤料液罐中加入第一洗滤液，使其通过所述双膜联用一体过滤系统，获得浓缩液。
  7. 根据权利要求6所述的方法，其中所述第一洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。
  8. 根据权利要求6所述的方法，其中所加入的第一洗滤液的体积为微滤截留液体积的1～5倍。
  9. 根据权利要求1所述的方法，其中将步骤(ii)中所获得的超滤滤过液做为第一洗滤液加入至所述微滤料液罐中，使所述超滤滤过液通过所述双膜联用一体过滤系统，获得浓缩液。
  10. 根据前述权利要求中任一项所述的方法，其中向步骤(ii)中所获得的含有口蹄疫病毒的浓缩液中加入第二洗滤液，使所述浓缩液中的小分子杂质透过超滤膜，获得纯化的病毒浓缩液。
  11. 根据权利要求10所述的方法，其中所述第二洗滤液为pH7.2～pH9、电导5-300mS/cm的缓冲溶液。
  12. 根据权利要求10所述的方法，其中所加入的第二洗滤液的体积为所述病毒浓缩液体积的1～10倍。
  13. 根据前述权利要求中任一项所述的方法，其中所述微滤装置与第一恒流泵连接、所述超滤装置与第二恒流泵连接以动态控制过膜通量。
  14. 根据权利要求13所述的方法，其中所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端。
  15. 根据权利要求13或14所述的方法，其中所述过膜通量为微滤膜或超滤膜的临界膜通量的65％～100％。
  16. 根据权利要求15所述的方法，其中所述过膜通量为10-150LMH。
  17. 根据前述权利要求中任一项所述的方法，其中所述微滤膜和所述超滤膜的剪切速率各为1500～4000s-1。
  18. 根据前述权利要求中任一项所述的方法，其中所述微滤膜和所述超滤膜的物料与膜面积比均各为10～500L/m2。
  19. 根据权利要求1所述的方法，其中在步骤(iii)之后对所述含有口蹄疫病毒的浓缩液进行灭活。
  20. 根据权利要求19所述的方法，其中在所述灭活之后对所述含有口蹄疫病毒的浓缩液进行乳化。
  21. 根据权利要求19或20所述的方法，其中在所述灭活之前或之后对所述含有口蹄疫病毒的浓缩液进行进一步纯化。
  22. 根据权利要求21所述的方法，其中所述进一步纯化通过PEG沉淀法或色谱层析法进行。
  23. 权利要求22所述的方法，其中所述色谱层析法包括排阻色谱层析法、离子交换色谱层析法、疏水色谱层析法和亲和色谱层析法。
  24. 根据前述权利要求中任一项所述的方法，其中在步骤(iii)之后对所述微滤装置和所述超滤装置进行清洗再生。
  25. 根据权利要求23所述的方法，其中所述清洗再生包括如下步骤：
    a)将纯水分别加入所述微滤装置和所述超滤装置以分别清洗其中的微滤膜和超滤膜，清洗过程中膜剪切速率提升至8000s-1～16000s-1，过膜通量控制在10～500LMH；
    b)经步骤a)清洗后将0.1～0.5M NaOH溶液分别加入所述微滤装置和所述超滤装置做进一步清洗，溶液温度为45～55℃，膜剪切速率为8000s-1～16000s-1，过膜通量控制在50～500LMH，时间为30～60分钟；
    c)经步骤b)清洗后用纯水分别冲洗所述微滤装置和所述超滤装置至微滤滤过液和超滤滤过液的pH值降至9以下。
  26. 根据前述权利要求中任一项所述的方法，其中所述口蹄疫病毒毒株包括一种或多种口蹄疫病毒血清型毒株。
  27. 根据权利要求26所述的方法，其中所述血清型为O型、A型、C型、SAT1型、SAT2型、SAT3型或Asia1型。
  28. 根据前述权利要求中任一项所述的方法制备得到的口蹄疫疫苗。
  29. 根据权利要求28所述的口蹄疫疫苗在制备用于预防动物口蹄疫疾病的药物中的用途。
  30. 一种用于制备口蹄疫疫苗的装置，其包括双膜联用一体过滤系统，所述双膜联用一体过滤系统包括并行设置的微滤装置和超滤装置，所述微滤装置包括微滤料液罐、第一主泵和微滤膜，所述超滤装置包括浓缩罐、第二主泵和超滤膜；其中所述微滤装置用于除去所述口蹄疫病毒细胞培养液中的大颗粒杂质，所述超滤装置用于除去所述口蹄疫病毒细胞培养液中的小分子杂质；所述微滤装置和所述超滤装置同时运行对所述口蹄疫病毒细胞培养液同时进行微滤和超滤，从而在所述浓缩罐中形成含有口蹄疫病毒的浓缩液。
  31. 根据权利要求30所述的装置，其中所述微滤装置和所述超滤装置相互连接，以使得微滤滤过液和超滤截留液均置于所述浓缩罐中形成所述含有口蹄疫病毒的浓缩液。
  32. 根据权利要求30或31所述的装置，其中所述双膜联用一体过滤系统还包括第一恒流泵和第二恒流泵，所述微滤装置与所述第一恒流泵连接，所述超滤装置与所述第二恒流泵连接以动态控制过膜通量。
  33. 根据权利要求32所述的装置，其中所述第一恒流泵设置在所述微滤装置的滤过端，所述第二恒流泵设置在所述超滤装置的滤过端。
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