CN104491855B - Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof - Google Patents
Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof Download PDFInfo
- Publication number
- CN104491855B CN104491855B CN201410643314.3A CN201410643314A CN104491855B CN 104491855 B CN104491855 B CN 104491855B CN 201410643314 A CN201410643314 A CN 201410643314A CN 104491855 B CN104491855 B CN 104491855B
- Authority
- CN
- China
- Prior art keywords
- foot
- mouth disease
- virus
- disease virus
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof. Described method comprises: a) results virus liquid; B) deep layer membrane filtration, ultrafiltration, nucleic acid enzymolysis; C) strong anion exchange absorbing bed or adsorbed film purifying; D) PEG precipitation, chloroform isoamyl alcohol extracting; E) deactivation; F) SDGC purifying; G) ultrafiltration dialysis, aseptic filtration; H) stoste deposit or emulsification preparation. Aftosa whole virus particles marker vaccine antigen provided by the invention is the foot and mouth disease virus particle that homogeneous is complete, implanter body can be distinguished zoogenetic infection and immunity completely, not containing foot and mouth disease virus non-structural protein and other virions, not containing animal derived foreign protein, polypeptide, oligopeptides, effectively reduce vaccine injection and cause the potential allergic reaction of animal, carcinogenic and cause that zoonosis, as the potential risk of rabid ox disease, does not affect animal foodstuff safety and trade.
Description
Technical field
The present invention relates to preparation method of a kind of aftosa whole virus particles marker vaccine and products thereof, particularly onePrepare method of high yield, high-purity, high safe aftosa whole virus particles marker vaccine and products thereof on a large scale, the invention belongs toIn medical biotechnology field.
Background technology
Aftosa (Foot-and-mouthdisease, FMD) is by foot and mouth disease virus (Foot-and-mouthDiseasevirus, FMDV) the strong contagious disease of artiodactyl that causes, main harm ox, sheep, pig, camel etc., send outSick rate is high, and spread speed is exceedingly fast, and OIE is classified as category-A Amphixenosis first place. Although this disease is deadThe rate of dying not high (except cub), but can cause breeding performonce fo animals decline, and slaughter animal need spend a large amount of human and material resources andFinancial resources, also can affect international trade, cause serious economic loss and bio-terrorism.
Foot and mouth disease virus belongs to Picornaviridae, Hostis. At present known have 7 serotypes, A, O, C,SAT1, SAT2, SAT3, ASIA1, each serotype has again a lot of hypotypes, and the hereditary variation between different strains can cause antigenicityDifference. Popular 3 serotypes of China and pathogenicity are the strongest, distribution is the most extensive, are respectively A type, O type and ASIA1 type.
Aftosa non-structural protein L in purifying aftosa vaccine formula, B2, C3, D3, A3, AB123,3ABC content are very micro-,Containing the complete foot and mouth disease virus 146S of highly purified deactivation particle, do not cause that animal produces the antibody of anti-non-structural protein, naturally senseDye animal and produce non-structural protein antibody, the antibody that therefore detects 3AB1 in animal body or 3ABC non-structural protein exist withNo, can distinguish the immunity of animal foot and mouth disease virus and infect, immunity and the detection effect of the prevention and control of performance aftosa.
Vaccine inoculation is the effective means of specificity prevention FMD, and preparing vaccine is safely and effectively successfully to prevent, controlAnd even finally eliminate the prerequisite of FMD. FMD inactivated vaccine has good immunogenicity, in the process of prevention and control FMDIn play an important role, but some host cell residual proteins, nucleic acid, cow's serum in vaccine preparation process neutralized productIf albumin, chemical reagent etc. are not controlled, immune animal causes that the serious side reaction of animal is even dead, particularly canCan make animal carcinogenic or affect the food security of meat animal.
Cultivate virus with suitable host cell and prepare vaccine antigen or reconstituted drug, consider from drug safety, necessaryPurifying antigen is removed the impurity that affects foot and mouth disease virus vaccine security, so that it meets drug quality specification and safety requirements,Reduce in technique or raw and auxiliary material simultaneously as far as possible may be potential affect vaccine effect and safe material, particularly avoid as far as possibleUse animal derived raw and auxiliary material, reduce and propagate animal epidemic as infectious disease wind such as aftosa, blue otopathy, rabid ox disease, swine feversDanger.
Produce foot-and-mouth disease virus antigen with bioreactor and improved cell density, viral antigen output, increased simultaneouslyThe amount of cell impurity and additive, step or the technique of also increase separation, concentrated and purified foot and mouth disease virus. Past is because aftosaAntigenic storehouse set up and separation of biopolymer technical development degree not high, purifying foot and mouth disease virus mostly be ammonium sulfate etc. concentrate, pureChange technology. Modern scale is concentrated, purified virus antigen technology, in column chromatography, sucrose gradient ultracentrifugation technology, slipstreamHollow fiber post, in-depth filtration module are not almost used in live vaccine is produced, and the application of these technology and equipments is easy to epidemic diseaseSeedling quality control and guarantee, GMP certification and process certification.
It is vaccine antigen production matrix that existing production deactivation foot-and-mouth disease virus antigen adopts BHK-21 cell more, adopts and suspendsCultivate follow-up deactivation, concentration technology reach partial purification, emulsification make vaccine for. Although the immune effect of this vaccine reachesTo the requirement of vaccine code, but by the security requirement of modern vaccination, impurity content is high, total protein content is high, Effective Antigens 146SGranule content is low, to Effective Antigens amount, BHK-21 cell rests DNA, the thin residual protein of BHK-21, bovine serum albumin(BSA), deactivationAgent, anticorrisive agent and production process chemical additive do not clearly define and quantitatively, although within 2013, carried out new vaccine matterAmount standard, but the quality standard of vaccine and technique should improve constantly, ensureing that vaccine is stable, safety, effectively.
Summary of the invention
The object of the present invention is to provide a kind of extensive preparation high yield, high-purity, high safe aftosa totivirusMethod of grain marker vaccine and products thereof.
The aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield of the present invention, high-purity, high safetyMethod, it is characterized in that comprising the following steps:
A) 100000L bioreactor entirely suspends and cultivates BHK-21 cell, and cell density reaches 3-5 × 106When/milliliter, pressAccording to virus infections plural number MOI0.01-0.1 inoculation foot and mouth disease virus cell adapted strain, prepare virus stock solution used, mixing speed is no more than40rpm, cultivates 4 days results virus liquids, uses the centrifugal cell fragment of removing of preparative low speed continuous flow centrifuge, simultaneously in resultsClear liquid and precipitation; Be deposited in Triton-X-100 exist condition under cracking Foot-and-mouth disease virus Infectious Cycle and cell membrane fragments,The final concentration of described Triton-X-100 is 0.1-0.2%, after multigelation 3 times ultrasonic 3 times, with preparative low speed Continuous Flow fromThe centrifugal collection supernatant of scheming, merges supernatant; The quality control index of this step is: foot and mouth disease virus titre for >=107logTCID50/ mL, total protein concentration is 5-7 gram, foot and mouth disease virus 146S content is 70-85 μ g/ml, nonstructural protein 3A BCResidual volume≤25ng/ml, bovine serum albumin(BSA) residual volume≤100ng/ml, BHK-21 cell rests protein content≤100ng/ml,BHK-21 cell rests DNA amount≤5000pg/ml, Triton-X-100 residual volume≤6000 μ g/ml, endotoxin≤150EU/ milliRise;
B) film in-depth filtration, ultrafiltration, nucleic acid enzymolysis:
The viral supernatant that step a) obtains, successively through the filter membrane in-depth filtration of 0.8 μ m, 0.45 μ m, 0.22 μ m with cutThe value of staying is 100,000-300,10 times of ultrafiltration of the film bag of 000MWCO or hollow fiber column; High special, highly active nuclease are pressed20-50×103Units per liter joins in the supernatant after filtration, 2-8 DEG C of effect 9-18 hour; The quality control of this step refers toBe designated as: foot and mouth disease virus titre is 107.0-108.0logTCID50/ mL, total protein concentration is 6-6.5 gram, foot and mouth disease virus 146S containsAmount is 50-60 μ g/ml, nonstructural protein 3A BC residual volume≤7ng/ml, bovine serum albumin(BSA) residual volume≤30ng/ml, BHK-21 cell rests protein content≤90ng/ml, BHK-21 cell rests DNA amount≤4500pg/ml, Triton-X-100 residual volume≤510 μ g/ml, endotoxin≤500EU/ milliliter;
C) strong anion exchange absorbing bed or adsorbed film purifying:
Through b) the viral supernatant after enzymolysis of step, through 0.22 μ m membrane filtration, then by reinforcing yin essence ion-exchange adsorbed filmOr adsorbent bed, carry out stepwise elution; The quality control index of this step is: foot and mouth disease virus titre is 107.0-108.0logTCID50/ mL, total protein concentration is 2-3 gram, foot and mouth disease virus 146S content is 30-40 μ g/ml, nonstructural protein 3A BCResidual volume≤5ng/ml, bovine serum albumin(BSA) residual volume≤75ng/ml, BHK-21 cell rests protein content≤50ng/ml, BHK-21 cell rests DNA amount≤2500pg/ml, Triton-X-100 residual volume≤850 μ g/ml;
D) PEG precipitation, chloroform isoamyl alcohol extracting;
Through step c) viral supernatant after treatment precipitate with PEG, the resuspended precipitation of buffer solution, viral resuspended liquid is through containing extractingLiquid extracting, described extract obtains after chloroform and isoamyl alcohol are mixed by 24: 1 volume ratios;
The quality control index of this step is: foot and mouth disease virus titre is 107.0-108.0logTCID50/ mL, total protein concentrationFor 3-4.2 gram, foot and mouth disease virus 146S content is 50-60 μ g/ml, nonstructural protein 3A BC residual volume≤15ng/ml, cow's serumAlbumin residual volume≤90ng/ml, BHK-21 cell rests protein content≤60ng/ml, BHK-21 cell rests DNA amount≤3500pg/ml, Triton-X-100 residual volume≤400 μ g/ml, PEG residual volume≤250 μ g/ml, chloroform residual volume≤350 μ g/ml, endotoxin≤55EU/ milliliter;
E) deactivation;
0.025% divinyl imines, 4 DEG C of deactivations 48 hours or 37 DEG C of deactivations 2 hours;
F) SDGC purifying;
Step deactivation liquid e) is by Continuous Flow 60% sucrose isodensity gradient ultracentrifugation purifying, this step quality controlIndex is: total protein concentration 2-2.5 gram, and foot and mouth disease virus 146S content is 20-30 μ g/ml, nonstructural protein 3A BC residual volume≤4ng/ml, bovine serum albumin(BSA) residual volume≤50ng/ml, BHK-21 cell rests protein content≤60ng/ml, BHK-21 cell is residualRemaining DNA amount≤2000pg/ml, Triton-X-100 residual volume≤250 μ g/ml, PEG residual volume≤200 μ g/ml, chloroformResidual volume≤400 μ g/ml, endotoxin≤800EU/ milliliter;
G) ultrafiltration dialysis, aseptic filtration
Through step f) virus liquid after purifying through ultrafiltration dialysis concentrated 25-50 doubly, virus liquid is through 0.22 μ m film degermingFilter, this step quality control index is: total protein concentration 2-2.5 gram, foot and mouth disease virus 146S content is 20-30 μ g/ml, non-structureAlbumen 3ABC residual volume≤3ng/ml, bovine serum albumin(BSA) residual volume≤50ng/ml, BHK-21 cell rests protein content≤30ng/ml, BHK-21 cell rests DNA amount≤100pg/ml, chloroform residual quantity≤20 μ g/ml, PEG residual volume≤10 μG/ml, TritonX-100 residual volume≤15 μ g/ml, endotoxin≤35EU/ milliliter;
H) stoste deposit or emulsification preparation.
Sucrose gradient ultracentrifugation is the standard method of virion or antigen purification all the time, is widely used in mad dogIn the technique of disease, hepatitis A, encephalitis, influenza vaccines, due to scale preparative chromatography, and the development of film thickening filtration technology, workIndustry chromatographic technique, ultracentrifugation technology, film ultrafiltration concentration technology are used in some traditional inactivated vaccines, so on the one handBe beneficial to foundation, optimization and the checking of scale technique, facilitated on the other hand the raising of product quality and quantity, even now byIn the particularity of foot and mouth disease virus and vaccine, above-mentioned production viral antigen method, technique are difficult to separation and purification and are adsorbed on hostVirus in cell membrane system, the output of raising cell culture and virus, obtain highly purified foot-and-mouth disease virus antigen, removes vaccineCell fragment, cell protein and nucleic acid in stoste.
Adopt conventional polyethylene glycerine (PEG) precipitation to concentrate and purifying, the impurity in vaccinogen liquid can only be removed a part.
Method provided by the invention is rationally used detergent TritonX-100 in whole process, concentration 0.1-0.2%,Make foot and mouth disease virus not produce aggegation or polymer or be adsorbed on other albumen and impurity, improved intact virus yield.
In method of the present invention, preferred, in the stepwise elution of step described in c), washing lotion is for containing 90mM chlorineChange the 4.7mM sodium phosphate buffer of pH6.5-7.0 of sodium, with pH7.5 containing the sodium phosphate of the 120mM of 0.35M sodium chloride andThe buffer solution stepwise elution of 1mMEDTA, every 1000 mL media are processed the virus liquid of 40-50ml.
In method of the present invention, preferred, steps d) described in PEG be PEG-8000 or PEG-6000, warpThe virus liquid of nuclease digestion, chromatographic purifying wash-out precipitates with PEG, makes the concentration 2.7-10% (W/V) of PEG in virus liquid,Sodium chloride concentration is 0.35-1M, 2-8 DEG C of vigorous stirring 1 hour, and preparative centrifuge centrifugal 10 minutes with 1000g, goesClearly, with containing 120mM sodium chloride, 6.2mM sodium phosphate, the resuspended precipitation of 1mMEDTA (pH7.5) buffer solution, volume is virus-culturing fluid10-20%.
In method of the present invention, preferred, step f) middle deactivation liquid is passed through Continuous Flow 60% sucrose isodensity ladderDegree ultracentrifugation purifying, centrifuge used is manufacture type series, large capacity rotary head volume is 3.2-8 liter, batch processing extracting2000 liters of liquid; Foot and mouth disease virus deactivation liquid adopts density gradient ultracentrifugation, and ultracentrifugation mode is Continuous Flow, and gradient is not for connectingContinuous gradient, comprises the gradient that (1) centrifugal force 36000-60000g forms; (2) gradient that centrifugal force 90000-120000g forms;(3) the flow velocity 10L/ hour of foot and mouth disease virus deactivation liquid; Level pad used is 0.04MPBS (pH7.2-7.6), 60%Sucrose solution adopts the 100mMNaCl that contains of pH7.6, the 0.04M phosphate buffer preparation of 0.1%Triton-X-100, rotorInject 1.6 liters of 60% sucrose solutions that prepare; With 20 ls/h of loadings, process foot and mouth disease virus PEG and precipitate resuspended liquid 2000Rise rotating speed 32000rpm.
In method of the present invention, preferred, step I) described in the adjuvant that adopts of emulsification be V201.
No matter BHK-21 cell suspends is entirely cultivated, microcarrier is cultivated, adhere-wall culture foot and mouth disease virus, has animal in culture mediumThe albumen of source property or enzyme, the remaining composition of host cell has DNA, nucleic acid, protein, the detergent in cultivation, purge process, poly-secondRemnants, the antibiotic remnants of alcohol, chloroform, inactivator, reached and reduced animal population allergic side reactions and animal length by purifyingPhase, inject potential carcinogenic risk in a large number. Foot-and-mouth disease virus antigen includes but not limited to foot and mouth disease virus A type, O type, sub-1 type, CType, SAT1-3 type and hypotype, topological type.
In vaccine, 146S antigen accurate quantification is 20-25 ug/ml, carries out rapid preparing seedling, overcomes the past according to bodyInterior potency test, by volume carries out the not science way of empirical training seedling, and antigen accurate quantification is in conjunction with long-term accumulation on the other handThis animal data can replace the challenge trial of this animal. Main is to reach immune effect and reduction allergic reaction in body.
Preparation method provided by the present invention easily controls and checking, and separation, method concentrated, purifying foot and mouth disease virus compriseThe methods such as chemical reagent, slipstream or hollow fiber column, centrifugal, chromatography are according to the Quality Control of vaccine technique and vaccine quality requirement, rightEach processing step is optimized and combination, and checking, forms technique. Current known scale is concentrated, partial purification is prepared aftosaThe method of antigen is to adopt PEG precipitation or chloroform, is characterized in that treating capacity is little, operation is difficult to industrialization and automation, mostly is peopleWork operation, can not control and pollute and quality control. The film adsorption property of foot and mouth disease virus has determined process using PEG in early stage in additionPrecipitation or chloroform are removed part foreign protein, nucleic acid, lipid, set up just pure antigenic storehouse, carry out deactivation, emulsification preparation mouthful with this antigenFever aphthous vaccine. Hydrophobic reactant, gel molecular sieve 2-3 step chromatography, or 2 step ultracentrifugation technology, and mostly be while analysis in a small amount and makeWith, in aftosa vaccine antigen is produced, scale is not used, and these method steps are many, and viral antigen yield is low, should not adviseModelling is produced and quality control, does not also meet drug's GMP and produces certification and technical process checking requirement.
The invention provides production process quality control index and method, particularly BHK-21 cell rests albumen, remnantsDNA content detects and additive residue detection is to use at live vaccine first by method of quality control checking, is Quality ControlMethod and the innovation of quality assurance aspect.
Foot-and-mouth disease virus antigen provided by the invention has been removed production process and has been used detergent, the enzyme of external source, cow's serum eggIn vain, host cell residual protein, host cell residual DNA, residue chemistry additive, is easy to foot and mouth disease virus vaccine quality controlHigh-volume produce with the scale of technique, strengthened the quality control of vaccine technique. High by the vaccine antigen purity of the method production,Clinical use side reaction rate is low.
Two of object of the present invention is to provide the aftosa totivirus preparing according to the method described in above any oneParticle marker vaccine.
In the present invention, preferred, middle Tot Prot 100 ug/ml in every milliliter of vaccine, foot and mouth disease virus 146S containsAmount 20-25 ug/ml, endotoxin content≤50EU/ milliliter, BHK-21 cell protein residual quantity≤30 nanograms/milliliter, BHK-21 cell DNA residual quantity≤100 pg/ml, bovine serum protein residual content≤50 nanograms/milliliter, chloroform residual quantity≤ 20 ug/ml, PEG residual volume≤10 ug/ml, divinyl imines remnants≤5 ug/ml, TritonX-100Remnants≤15 ug/ml, antibiotic remnants≤50 nanograms/milliliter, nonstructural protein 3A BC remnants≤5 nanograms/milliliter.
In aftosa whole virus particles marker vaccine provided by the invention, foot and mouth disease virus is homogeneous complete virion(146S), effect is high, and purity is high, not containing the non-structural protein of aftosa in Virus culture and jejune virion, noContaining animal derived foreign protein, polypeptide, oligopeptides. Effectively reduce vaccine injection and cause the potential allergic reaction of animal, carcinogenicRisk, has reduced and has caused that zoonosis, as the potential risk of rabid ox disease, aftosa, distinguishes zoogenetic infection and immunity completely, rightAnimal foodstuff safety and trade do not affect.
Aftosa non-structural protein L, B2, C3, D3, A3, AB123,3ABC in aftosa marker vaccine provided by the inventionContent is very micro-, containing the complete foot and mouth disease virus 146S of highly purified deactivation particle, injects animal body and produces aftosa structural proteinsAntibody or neutralizing antibody, make animal produce stronger humoral immunity, detects 3AB123 or 3ABC non-structural protein in animal bodyAntibody, can distinguish animal foot and mouth disease virus immunity and infect, become the mark epidemic disease of a kind of high safety, high-titer, high yieldSeedling, effective immunity and monitoring effect in performance prevention and control.
The adjuvant emulsion that marker vaccine provided by the invention is preferably W/O/W forms, and can strengthen artiodactyls pairComprehensive immunity of foot and mouth disease virus, strengthens neutralizing antibody and generates, strengthens cellular immunity.
The invention provides a kind of extensive preparation high yield, high-purity, high safe aftosa whole virus particles mark epidemic diseaseMethod of seedling and products thereof relates to virology, immunology, vaccinology and process, particularly marker vaccine, component withAnd method for making comprises purifying process, method of quality control and the index of scale foot and mouth disease virus. Method for making comprises that bioreactor is completeSuspension free serum culture BHK-21 cell and foot and mouth disease virus vaccine seeding are criticized virus, through multigelation, cracking or superSound, nucleic acid enzymolysis, in-depth filtration, reinforcing yin essence ion friendship adsorbent bed bed or film, deactivation, PEG precipitation, chloroform extract, sucrose density ladderDegree centrifugal purification, ultrafiltration dialysis, aseptic filtration, dilution emulsification preparation, can be used for commercial BHK-21 and adapt to or attenuated strain aftosaThe purifying of seedling, is also included within the various or hypotype foot and mouth disease virus that other sensitive cell lines are bred. BHK-21 cells is mainly doneFor the foot and mouth disease virus labelled antigen preparation of the various or hypotype of aftosa vaccine production matrix.
The invention provides from the method for the separation of BHK-21 host cell impurity, purifying foot and mouth disease virus particle, step bagDraw together 1) 100000L bioreactor culture infection cell; 2) process cell culture supernatant with nuclease and decomposition agent; Dissolve mouthFever aphthous virus infected cell, comprises employing decomposition agent; 3) adopt deep layer membrane filtration, ultrafiltration combination, reinforcing yin essence ion exchange bed or filmRemove a large amount of small molecular protein impurity and nucleic acid impurity; Control can scale operational volume; 4) adopt PEG and chloroform groupClose remove virus stock solution used lipid, separate and be adsorbed on cell membrane or the foot and mouth disease virus of polymerization; 5) be further purified the company of employingAfterflow SDGC; Invention provides purifying intact foot and mouth disease virus method, that this invention provides is complete, stable,Highly purified, there is good antigenicity, immunogenic foot-and-mouth disease virus antigen. Aseptic, GMP condition that existing invention also providesLower purifying has high activity, stable, highly purified foot and mouth disease virus industrialized preparing process.
Produce vaccine with passage cell, its component should, without to people and harmful albumen and the nucleic acid of healthy animal, should not produceBad side reaction, exogenous factor bacterium, fungi, mycoplasma, virus, parasite, rabid ox disease potential in reply production process are formerControlled. Deactivation purifying mark aftosa vaccine there is no quality standard or code both at home and abroad at present.
In the present invention, by purifying, host cell BHK-21 albumen≤30 nanograms/milliliter in vaccine, BHK-21 is thinBorn of the same parents DNA residual quantity≤100 pg/ml, safer to animal. Consider risk that exogenous factor in production process pollutes andThe security risk that some chemical substance additives are remaining possible, invention provides a large amount of safety verification methods. Epidemic disease is also providedThe key of seedling production process is accused point.
BHK-21 cell is the matrix that aftosa vaccine is produced, and passage cell is produced to vaccine, OIEOIE and domestic veterinary drug Quality Control department do not have strict code, and invention is with reference to the WHO of the World Health Organization, united states drug food supervision pipeThe pharmacopeia requirement of reason administration, China, further improves the purifying process of aftosa vaccine, has strengthened technique and quality control method is testedCard, meets international standards and has set up company standard from vaccine index. Tot Prot 100 ug/ml, foot and mouth disease virus146S content 20-25 ug/ml, endotoxin content≤50EU/ milliliter, BHK-21 cell protein residual quantity≤30 nanogram/in the leastLiter, BHK-21 cell DNA residual quantity≤100 pg/ml, bovine serum protein residual content≤50 nanograms/milliliter, chloroformResidual quantity≤20 ug/ml, PEG residual volume≤10 ug/ml, divinyl imines remnants≤5 ug/ml, TritonX-100 remnants≤15 ug/ml, antibiotic remnants≤50 nanograms/milliliter, nonstructural protein 3A BC remnants≤5 nanogram/in the leastRise.
In the present invention, provide the aftosa marker vaccine preparation method of scale continued operation, improved density region bandCentrifugal, tangential flow filtration, ultrafiltration, ion-exchange absorption bed.
Current domestic foot and mouth disease virus purifying mainly adopts gel chromatography, and centrifugal and hyperfiltration process for large-scale production is solidifyingIt is little that glue-line is analysed applied sample amount, how to use in laboratory, can not meet quality and the safety requirements of passage cell vaccine, both at home and abroad listingThe vaccine host cell residual DNA using is not more than 10 nanograms/agent or is not more than 100 piks/agent, according to aftosa marker vaccinePrinciple, in vaccine, aftosa structural proteins require, for intact virus, almost there is no defective virus, non-structural protein, for meeting thisStandard, needs new technology and new method. The method and the Quality Control that in the present invention, provide host cell BHK-21 to remove, adopt sucroseThe isopycnic banding heart, PEG precipitation, chloroform extracting, ion-exchange adsorbed film. Chromatography molecular sieve gel chromatography is used for the egg of recombinatingViral vaccine in vain. Ion-exchange adsorbed film or post bed are removed DNA in a large number.
Benzonase nuclease is processed a large amount of large molecular dnas that reduce in virus stock solution used, further reduces the carcinogenic of vaccineRisk. Method in invention has overcome the bottleneck of current known aftosa vaccine production technology, can produce high activity, highly purifiedFoot-and-mouth disease virus antigen, in output, process time, purity, be better than known foot and mouth disease virus purified vaccine method, as thoroughlyAnalyse, sucrose pad is centrifugal, sieve chromatography and non-specific ion-exchange chromatography.
Provide that purifying intact whole virus particles is stable, high yield, highly purified preparation method. Input/output is providedThe technique of successful and method.
Dissolve host cell
Because foot and mouth disease virus breeds in BHK-21 cell, the cell that formed cytopathy cracking, part virus discharges,Part virus is in cell or on cell fragment, and the present invention adopts continuous low speed centrifuge to collect viral supernatant, and precipitation is carried outAfter ultrasonic, freezing-thawing and cracking, carry out again low-speed centrifugal, merge 2 part supernatants. There are many methods as freeze thawing, height ooze, ultrasonic, decomposition agentCracking solubilized cell, discharge foot and mouth disease virus, but from the convenience of large-scale production be with considering, take freeze thawing, ultrasonic, splitSeparate agent dissolved cell, improve foot and mouth disease virus output.
Decomposition agent
According to the present invention, almost do not use decomposition agent except in culture process, the step of purification and separation is used decomposition agent,Choose pharmacy, veterinary science and think safe surfactant TritonX-100, concentration is 0.1-0.2%.
Nuclease
Many nucleases use in laboratory, as Benzonase, Pulmozyme or other DNA enzymes, nuclease, but considerThe safety thought of veterinary science, pharmacy, the present invention selects Benzonase, can pass through phosphate bond fission hydrolytic nucleic acid. Use denseDegree 1-100units/ml, invention provide aftosa nutrient solution in cracking and purge process, by add nuclease and non-fromSub-surface activating agent strengthens the removal method of aftosa impurity; Preferably, nuclease is Benzonase;
In-depth filtration
In technique of the present invention, lysate and virus-culturing fluid need to filter clarification, remove cell fragment and a part of assortedMatter, many industrialized filter membranes and filter are available, comprise in-depth filtration or end-filtration, the invention provides aftosa deep layerThe combination of filtration or membrane filtration compares its advantage in example, selects 0.8 μ m, 0.45 μ m, 0.22 μ m film.
Ultrafiltration and dialysis
In example of the present invention, foot and mouth disease virus nutrient solution, stoste, refined solution at least need dialysis, remove little molecule assortedMatter and chemical substance are residual, also need to control feasible operational volume, and the step of dialysis and ultrafiltration concentration is provided in invention, makeFoot and mouth disease virus reaches partial purification and concentrated, and the ultrafiltration in invention preferentially adopts the Hollow Fiber Ultrafiltration post of slipstream, ultrafiltrationThe cutoff value in the aperture of film is 100,000-300, and 000MWCO embodies and has foot and mouth disease virus than conventional film bag in exampleShear little, the damaged few advantage of virus.
Remove the sugar in solution in production process, salt, on-liquid solvent, low-molecular material by ultrafiltration dialysis and buffer-exchangedMatter, changes and stablize particle and the pH environment of foot and mouth disease virus, the present invention in the processing step of gathering in the crops after cracking at purification stepBefore switching, before vaccine preparation, all use ultrafiltration, adjust buffer solution, adjust volume. Concentrating virus liquid is applicable to carrying of subsequent purificationAmount requirement.
Highly purified
According to invention marker vaccine, single chromatography and Continuous Flow ultracentrifugation can not meet aftosa marker vaccineQuality requirement and standard, sieve chromatography as disclosed in most patents, obtain in a small amount the method for foot and mouth disease virus 146S. CounterpartIn fever aphthous virus liquid, remove DNA effect, the ion-exchange of reinforcing yin essence ion is applicable to the processing of a large amount of virus stock solution useds.
In example of the present invention, a kind of strong anion exchange absorbing bed or film are adopted at foot and mouth disease virus purifying, by the moonAfter ion-exchange, remove a large amount of nucleic acid, and concentrated virus stock solution used. In invention example, hand over QSepharoseXL ionChange adsorbent bed or chromatography, reached the object of purifying foot and mouth disease virus. Because the sieve chromatography carrying capacity limit is column volume20%, and separating effect dependent protein concentration, limit the use of sieve chromatography in aftosa vaccine suitability for industrialized production. ThisStrong (iii) yield of fast (ii) binding ability of anion-exchange chromatography (i) flow velocity of bright selection is high, (iv) does not need to install and clean testingCard (v) is made filter core, there is no service life and the safe worry of storage; Anion exchange filter core purifying hoof-and-mouth disease in examplePoison is better than ion exchange column, and some aftosa structural proteins separate with complete aftosa structural proteins, reach marker vaccine qualityPart requirement. This effect is also innovation of the present invention. Therefore the invention provides aftosa structure, non-structural protein andAftosa complete virion 146S separates, the method for purifying, also to the open method of aftosa viral purification PatentsImprove.
In the present invention, there are many steps need to change buffer solution, the one, directly add the solution of preparation as nuclease, TritonX-100, PEG-80000, chloroform, combine and optimize its use in invention, ensured that foot and mouth disease virus is not solidifyingPoly-, consistency, have stronger centrifugation to lipid, other impurity of nucleic acid. Invent on the other hand, before consummate stepMultiplex hydrophobic filter DuraporePVDF filter (MillipacfromMillipore) or Sartopore2 filter afterwardsDevice, aperture, at 0.8 micron, 0.45 micron or 0.22 micron, is removed partial impurities and changes liquid.
Using high level salt solution to remove DBP is an aspect of of the present present invention, is also applicable to other virus type purified vaccines,Therefore use filter or hollow fiber column ultrafilter or the filtration of different pore size before ion-exchange chromatography, wash with 1MNaCl at leastThe DBP of de-ion exchange bed. The purification effect of the salt pair aftosa of variable concentrations embodies in example.
Compared to prior art, the invention has the advantages that:
1. total protein content in marker vaccine antigen, effective complete labelled antigen content, BHK-21 cell rests are providedDNA, remaining bovine serum albumin(BSA), remaining BHK-21 cell protein content, remaining BHK-21 cell DNA content, remaining divinylImines, remaining PVOH 6000 content, remaining Determination of Trichloro Methane,, make small-molecule substance or chemical substance as PEG chloroform etc.Be down to minimumly, ensured efficient, the mark, safety of vaccine, security thereby the guarantee animal food safety of stable and animalSafety with the mankind.
2. not containing ectogenic albumen, polypeptide, oligopeptides, use if added, all removed in a large number, reduced animal epidemic diseaseSick propagation risk.
3. the method that provides of invention comprises modern concentrated, ultrafiltration, purification technique combination, and technological process is reasonable, and aftosa is completeWhole viral yield is high and purity is high, is easy to large-scale production and quality control; The quality control method that invention provides is all ripe warpCross the quality inspection method of foundation and multiple authentication, accurate, sensitive, easy, quick.
Brief description of the drawings
Fig. 1 is that one of the present invention is prepared high yield, high-purity, high safe aftosa whole virus particles mark epidemic disease on a large scaleThe method flow diagram of seedling;
Fig. 2 is that one of the present invention is prepared high yield, high-purity, high safe aftosa whole virus particles mark epidemic disease on a large scaleThe method quality control method of seedling and Quality Control point.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be along with descriptionMore clear. But embodiment is only exemplary, scope of the present invention is not formed to any restriction. Those skilled in the art shouldThis understanding can be repaiied the details of technical solution of the present invention and form under without departing from the spirit and scope of the present inventionChange or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
The preparation of embodiment 1 high yield, high-purity aftosa whole virus particles marker vaccine
Comprise the following steps:
A) 100000L bioreactor entirely suspends and cultivates BHK-21 cell, and cell density reaches 3-5 × 106When/milliliter,According to virus infections plural number MOI0.01-0.1 inoculation foot and mouth disease virus cell adapted strain (swine foot-and-mouth disease virus/Mya98-XJ-2010 strains, Inner Mongolia Bigvet Biotechnology Co., Ltd.'s preparation), prepare virus stock solution used, mixing speed is no more than 40rpm,Cultivate 4 days results virus liquids, use the centrifugal cell fragment of removing of preparative low speed continuous flow centrifuge, results are upper cleer and peaceful heavy simultaneouslyForm sediment, be deposited in 0.2%Triton-X-100 exist condition under cracking Foot-and-mouth disease virus Infectious Cycle and cell membrane fragments, repeatedlyAfter freeze thawing 3 times, ultrasonic 3 times (peak power output 3 × 30s); With the centrifugal collection supernatant of preparative low speed continuous flow centrifuge,Merge supernatant, wherein foot and mouth disease virus titre is >=107.0logTCID50/mL；
B) nucleic acid enzymolysis;
Through step virus liquid a), successively through membrane filtration and the cutoff value 100 of 0.8 μ m, 0.45 μ m, 0.22 μ m,000-300,10 times of ultrafiltration of the film bag of 000MWCO or hollow fiber column;
High special, highly active nuclease Benzonase join employing virus cracking liquid by 20-50 × 103 units per literIn, 2-8 DEG C of effect 9-18 hour; Wherein foot and mouth disease virus titre is 107.0-108.0logTCID50/mL；
C) in-depth filtration and ultrafiltration, reinforcing yin essence ion-exchange;
Through step virus liquid b), through 0.22 μ m membrane filtration, by QSepharoseXL ion-exchange absorption bed or suctionMembrane, 1000 mL media are processed the virus liquid of 40-50ml, and washing lotion is the 4.7mM sodium phosphate buffer that contains 90mM sodium chloride(pH6.5-7.0), with the sodium phosphate of 120mM and the buffer solution of 1mMEDTA (pH7.5) stepwise elution containing 0.35M sodium chloride; ItsMiddle foot and mouth disease virus titre is 107.0-108.0logTCID50/mL；
Employing virus cracking liquid has been removed the material of little molecular weight after filtration with ultrafiltration, then through the macromolecular virus of nuclease digestionRNA and cell RNA, as a rule, anion exchange resin is removed host cell DNA or the nucleic acid in virus liquid, oneA little anion media include but not limited to DEAE cellulose, DEAE agarose, DEAE biogum, deae dextran.
D) PEG precipitation, chloroform extract;
For step virus liquid c) PEG-8000 or PEG-6000 precipitation, PEG-8000 or PEG-6000 are made into 40%Storage liquid, 6.2mM sodium phosphate, 1mMEDTA buffer solution preparation 5M sodium chloride, in the present embodiment through nuclease digestion, chromatographic purifyingThe virus liquid of wash-out precipitates with PEG-8000, makes the concentration 4% (W/V) of PEG-8000 in virus liquid, and sodium chloride concentration exists100mM, after vigorous stirring 2-8 DEG C 1 hour, preparative centrifuge centrifugal 10 minutes with 1000g, removes supernatant, with containing 120mM chlorineChange sodium, 6.2mM sodium phosphate, the resuspended precipitation of 1mMEDTA (pH7.5) buffer solution, the 10-20% that volume is virus-culturing fluid.
Above-mentioned virus harvest liquid, as foot and mouth disease virus stoste, adds extract, and described extract is by by trichlorineMethane and isoamyl alcohol obtain after mixing by 24: 1 volume ratios, make a large amount of foot and mouth disease virus particles rest on water and water and milk circleFace. Virus stock solution used adds isopyknic extract, mix up and down 1 minute, by preparative with the centrifuge of multitube rotary head 20Under DEG C condition 3, centrifugal 10 minutes of 000rpm, results water, carries out 2 extractings to interface, merges water and obtains foot and mouth disease virusExtract. Wherein foot and mouth disease virus titre is 107.0-108.0logTCID50/mL；
E) deactivation;
0.025% divinyl imines, 4 DEG C of deactivations 48 hours or 37 DEG C of deactivations 2 hours
F) SDGC purifying;
Step stoste one step Continuous Flow sucrose isodensity gradient ultracentrifugation purifying e), centrifuge used is manufactureType series, large capacity rotary head volume is 3.2-8 liter, 2000 liters of batch processing extracts; Foot and mouth disease virus deactivation liquid adopts density level bandsDegree ultracentrifugation, ultracentrifugation mode is Continuous Flow, gradient is discontinuous gradient, comprises (1) centrifugal force 36000-60000g shapeThe gradient becoming; (2) gradient that centrifugal force 90000-120000g forms; (3) cumulative volume of saccharose gradient and aftosa deactivation liquid is8L, has the remnants in micro-BHK-12 cell impurity and leading portion step in deactivation liquid; (4) flow velocity of foot and mouth disease virus deactivation liquid10L/ hour; Step g) level pad used is 0.04MPBS (pH7.2-7.6), 60% sucrose solution buffer solution(0.04M phosphoric acid, 100mMNaCl, 0.1%Triton-X-100, pH7.6) preparation, rotor injects 1.6 liters of 60% sugarcanes that prepareSugar juice; With 20 ls/h of loadings, process foot and mouth disease virus PEG and precipitate 2000 liters of resuspended liquid, rotating speed 32000rpm;
G) ultrafiltration dialysis, dilution, aseptic filtration
Step f) stoste also concentrates 25-50 doubly through ultrafiltration dialysis, and the stoste after concentrating is through 0.22 μ m film aseptic filtration;
I) stoste deposit or emulsification preparation;
Stoste is laid in or through dilution emulsification preparation, the adjuvant that emulsification adopts is V201.
Foot and mouth disease virus marker vaccine large-scale production procedure figure and quality control method and Quality Control point as depicted in figs. 1 and 2, itsIn, in concrete foot and mouth disease virus marker vaccine large-scale production process, the quality control index system of each step is as shown in table 1 below.
The effect of embodiment 2. nucleic acid enzymolysis step
100000L bioreactor foot and mouth disease virus medium centrifugal precipitates through freeze thawing, ultrasonic, in technological operation, adds0.1%TritonX-100 (Sigma company product), merges supernatant and precipitation process liquid, adds Benzonase (MerckKgaA, 50units/ml) and MgCl2(2mM), effect 1 hour. Remove precipitation with continuous flow centrifugation. In-depth filtration adopts successively0.8 micron, 0.45 micron filter filters (German Sartorius company product), concentrates 5 times of use with 0.05 micron of hollow fiber column6 times of volume buffer solutions are containing 1.0MNaCl/50mMTris, and pH7.5 and 4 times of volume buffer solutions are containing 0.4MNaCl/50mMTrisLiquid, pH7.5. concentrate dialysate loading SepharoseQ-XL (Amersham) post, hoof-and-mouth disease venom buffer solution are changed in dialysisContaining 0.55MNaCl/50mMTris, pH7.5 wash-out and collection, collect liquid and further use sugarcane PEG/CH3Cl purifying, by examinationAgent box detects residual DNA.
TritonX-100/Benzonase processes the impurity content that makes finished product in conjunction with follow-up ion-exchange chromatography, purifyingReach the requirement of marker vaccine, multiple batches of DNA residual volume≤100pg/ agent. In processing step, there is no TritonX-100/Benzonase nuclease, end product BHK-21 cell DNA remnants are 20-80 times, reach 2-16 nanogram/agent. Triton is describedX-100/Benzonase has reduced BHK-21 cell DNA remnants in finished product aftosa stoste in a large number. TritonX-100 increases simultaneouslyStrong DNA discharges from cell, and Benzonase digestion 1 hour, has strengthened the degraded that is discharged into the DNA in nutrient solution.
In process optimization, we are from having tested various anionic exchange mediums, from QAE550C, SuperQ650M(Tosoh company product), QSepharoseHP, ANXSepharose4FF, DEAESepharose, QSepharoseXL, QSepharoseBigBead, QSepharoseFF (Amersham company product), although these media are applicable to virusPurifying, removes BHK-21 cell protein, DNA but Q-SepharoseXL is comparatively applicable to aftosa large-scale industrialization, and flow velocityHurry up, binding ability is strong, foot and mouth disease virus particle loss is less than 30%.
Sieve chromatography is as SephacrylS300, SephacrylS500Sepharose4FF, Sepharose6FF(allpurchasedfromAmersham company product), although separating on foot and mouth disease virus and other small molecular weight impuritiesSuccessful, but treating capacity is little, foot and mouth disease virus loss reaches 30-40%, concentrated therefore to technique virus liquid albumen requirement in early stageBased on above result, process flow steps is as Fig. 1 and Fig. 2.
Embodiment 3 buffer liq displacements or ultrafiltration, concentrated, dialysis
100000L bioreactor culture liquid is through centrifugal, freeze thawing, ultrasonic, centrifugation step, Benzonase nuclease(50units/mi) digestion 1 hour, 0.1%TritonX-100 effect 30 minutes, with 0.5 micron of filter, MillistakDE30/60 filter (purchase of Millipore company) clarification, clarified solution with same volume buffer solution containing (0.6MNaCl/50mMHEPES,PH7.5) be diluted to containing 0.3MNaCl 300kD filter (Biomax300, Pellicon2module, Millipore companyProduct) concentrated 10 times, with 2 times of volume dislysates contain (0.3MNaCl/50mMHEPESpH7), 2 times of volume dislysates contain(0.6MNaCl/50mMHEPESpH7.5), 2 times of volume dislysates (1.0MNaCl/50mMHEPESpH7.5), 3 times of bodiesLong-pending dislysate (0.3MNaCl/50mMHEPESpH7.5) is dialysed respectively, measures electric conductivity and protein content and finds NaCl saltConcentration is in the time of 0.6-0.8M, and 10-20KD protein stream is worn, and is applicable to dividing of hoof-and-mouth disease venom small molecular material and macromolecular substancesFrom.
The single filter method of embodiment 4 foot and mouth disease virus labelled antigen purifying
BHK-21 cell is in 1000000L bioreactor culture, cultivates after 3 days cell density and reaches 1,000,000 of 2-3/in the leastRise, with 0.01MOI infection cell, cultivate 3 days. Centrifuge results virus liquid, precipitation adds 0.1%TritonX-100 repeatedly 3Inferior freeze thawing, ultrasonic degradation, resuspended centrifugal, supernatant and virus liquid merge, and add Benzonase (50U/ml) 37 DEG C of digestion 10Minute, B/T harvest liquid detects by example 2 after in-depth filtration, membrane filtration. Hollow fiber column for clarified solution (0.05 micron pore size,20 centimetres of fiber length, 1.50 square metres of areas), with shear rate 2000s, 7 liters of/square metre of sample introductions, infiltration valve portion closesClose, produce permeable face pressure (inlet pressure 38kPa, outlet pressure P31kPa, osmotic pressure 17kPa), transmembrane pressure 17kPa is denseContracting 50-100 doubly, prepares buffer solution with the vaccine of 10 times of volume dislysates and 6 times of volumes and dialyses, filter used aperture 0.8,0.45,0.22 micron.
Foot and mouth disease virus purity and output detect and press Fig. 2 method, and aftosa intact virus yield rate is 69%, viral purificationIn liquid, host cell DNA residual volume is 10ng/ agent, contains 0.0135% TritonX-100, belongs to pharmacy tolerance interval.It is limited that data show that single ultrafiltration dialysis is removed marker vaccine impurity, need to be further purified or combine additive method.
Embodiment 5 aftosa vaccine purifying process and quality controls
Press Fig. 1, Fig. 2 flow process, protein determination method: 2005 editions annex 29VIB of Chinese pharmacopoeia, Residual exogenous DNA measuresDetermine method: 2005 editions annex 46IXB of Chinese pharmacopoeia, polyethylene glycol determination of residual amount method, 2005 editions annex 32VIG of Chinese pharmacopoeia, threeChloromethanes determination method, 2005 editions annex 35VIO of Chinese pharmacopoeia, antibiotic residual quantity determination method, 2005 editions annex of Chinese pharmacopoeia45IXA, 2005 editions annex 38VIID of determination of moisture method Chinese pharmacopoeia, free formaldehyde mensuration≤0.2g/L Chinese pharmacopoeia 2005Version annex 34VIL, molecular exclusion high pressure liquid chromatography (HPLC) on-line analysis vaccine antigen purity, online FMD virus extract, formerLiquid 100 microlitres, concentrated stoste 100 microlitres, refined solution 100 microlitres inject analytic type molecular exclusion chromatography post (TSK-GelPWcolumnG6000PW.sub.XL，particlesize17.mu.，poresize1000.ANG)(ToshoBiosciencesLLC.; Montgomeryville, Pa.), equilibrium liquid PBS (without Ca2+orMg2+) balance pillar, flow velocity1ml/ divides, ultraviolet absorption value 215nm, and peristaltic pump adopts Agilent1100TMSubsidiary ChemStationTMSoftware (AgilentTechnologiesInc.; PaloAlto, Calif.), BHK-21 cell rests DNA detects and adopts PicoGreenTMQuantitatively examinationAgent box (InvitrogenCorp.; Carlsbad, Calif.), lambdaDNA is as standard. BHK host cell proteins is measuredKit (CygnusTechnologies, Inc.4701SouthportSupplyRd.SE, Suite7Southport, NC28461USA) undertaken by operating instruction. Quality control in technique and assay are in table 1. The inspection of labelled antigen stock solution qualityThe results are shown in Table 2.
The main quality inspection result of table 2.3 batch labelled antigen semi-finished product
The quality standard of vaccine: foot and mouth disease virus 146S content 20 ug/ml, endotoxin content≤50EU/ milliliter,BHK-21 cell protein residual quantity≤30 nanograms/milliliter, BHK-21 cell DNA residual quantity≤100 pg/ml, ox blood are pureProtein residue amount≤50 nanograms/milliliter, chloroform residual quantity≤20 ug/ml, PEG residual volume≤10 ug/ml, twoAziridine remnants≤5 ug/ml, TritonX-100 remnants≤15 ug/ml, antibiotic remnants≤50 nanogram/in the leastRise nonstructural protein 3A BC remnants≤5 nanograms/milliliter.
The ability of embodiment 6 foot and mouth disease virus marker vaccine safety, effect and induction foot and mouth disease virus non-structural proteinTest
Prepare aftosa whole virus particles marker vaccine according to the method for embodiment 1, the antigen PBS preparing(pH7.6) be diluted to 3.5-10 microgram 146S/ milliliter, emulsification need adopt Seppic company by adjuvant V206: antigen=1: 1 is (heavyAmount: weight) mix, vaccine emulsification is specific as follows:
1) adjuvant and vaccinogen liquid respectively water-bath heat 31 ± 1 DEG C;
2) adjuvant is pressed 350rpm stirring, adds vaccinogen liquid and continue to stir 5 minutes in 5 seconds;
3) stopping being stirred in room temperature places 1 hour;
Safety test: animal ox, the various vaccine 8ml of the each 5 scalp hemostasis of pig, observe local and general reaction 10 days, andRecord.
Potency test: respectively choose the ox of the 12-18 month without foot-and-mouth disease antibody, be divided into 5 groups, every group of 6 ox muscle immunity 1ml epidemic diseaseSeedling, 3 oxen unavoidably in contrast, are observed 10 days, and the 30th day with containing 104The viral tongue intracutaneous of ID50 is attacked.
Non-structural protein antibody produces ability test: with containing each 5 of the vaccine injection ox of 3-7 ug/ml 146S and pig,Within after immunity 7,21 days, inject once more respectively, after one week of last immunity, with commercial foot and mouth disease virus ABC antibody test examinationAgent box detects 3ABC antibody.
Result: pig ox be not all checked through foot and mouth disease virus nonstructural protein 3A BC antibody. Therefore according to detection kitSensitivity and foot and mouth disease virus vaccine purity, aftosa marker vaccine contains at most 3ABC between 1-4ng, can be used as marker vaccineUse, pig and tri-injecting immunes of Niu Fanfu are not produced to antibody, the infection and immunity of difference infected pigs, ox. Marker vaccineSafety, effect, induction port fever aphthous non-structural protein antibody ability are in table 3.
Table 3
Claims (6)
1. a method for the aftosa whole virus particles marker vaccine of extensive preparation high yield, high-purity, high safety, its spyLevy and be to comprise the following steps:
A) 100000L bioreactor entirely suspends and cultivates BHK-21 cell, and cell density reaches 3-5 × 106In/when milliliter, is according to diseasePoison infection multiplicity MOI0.01-0.1 inoculation foot and mouth disease virus cell adapted strain, prepares virus stock solution used, and mixing speed is no more than40rpm, cultivates 4 days results virus liquids, uses the centrifugal cell fragment of removing of preparative low speed continuous flow centrifuge, simultaneously in resultsClear liquid and precipitation; Be deposited in Triton-X-100 exist condition under cracking Foot-and-mouth disease virus Infectious Cycle and cell membrane fragments,The final concentration of described Triton-X-100 is 0.1-0.2%, after multigelation 3 times ultrasonic 3 times, with preparative low speed Continuous Flow fromThe centrifugal collection supernatant of scheming, merges supernatant; The quality control index of this step is: foot and mouth disease virus titre for >=107logTCID50/ mL, total protein concentration is 5-7 gram, foot and mouth disease virus 146S content is 70-85 μ g/ml, nonstructural protein 3A BCResidual volume≤25ng/ml, bovine serum albumin(BSA) residual volume≤100ng/ml, BHK-21 cell rests protein content≤100ng/ml,BHK-21 cell rests DNA amount≤5000pg/ml, Triton-X-100 residual volume≤6000 μ g/ml, endotoxin≤150EU/ milliRise;
B) film in-depth filtration, ultrafiltration, nucleic acid enzymolysis:
The viral supernatant that step a) obtains, successively through filter membrane in-depth filtration and the cutoff value of 0.8 μ m, 0.45 μ m, 0.22 μ mBe 100,000-300,10 times of ultrafiltration of the film bag of 000MWCO or hollow fiber column; High special, highly active nuclease are pressed 20-50×103Units per liter joins in the supernatant after filtration, 2-8 DEG C of effect 9-18 hour; The quality control index of this stepFor: foot and mouth disease virus titre is 107.0-108.0logTCID50/ mL, total protein concentration is 6-6.5 gram, foot and mouth disease virus 146S contentFor 50-60 μ g/ml, nonstructural protein 3A BC residual volume≤7ng/ml, bovine serum albumin(BSA) residual volume≤30ng/ml, BHK-21Cell rests protein content≤90ng/ml, BHK-21 cell rests DNA amount≤4500pg/ml, Triton-X-100 residual volume≤510 μ g/ml, endotoxin≤500EU/ milliliter;
C) strong anion exchange absorbing bed or adsorbed film purifying:
Through b) the viral supernatant after enzymolysis of step, through 0.22 μ m membrane filtration, then by reinforcing yin essence ion-exchange adsorbed film or suctionAttached bed, carries out stepwise elution; The quality control index of this step is: foot and mouth disease virus titre is 107.0-108.0logTCID50/ML, total protein concentration is 2-3 gram, foot and mouth disease virus 146S content is 30-40 μ g/ml, nonstructural protein 3A BC residual volume≤5ng/Ml, bovine serum albumin(BSA) residual volume≤75ng/ml, BHK-21 cell rests protein content≤50ng/ml, BHK-21 cell restsDNA amount≤2500pg/ml, Triton-X-100 residual volume≤850 μ g/ml;
D) PEG precipitation, chloroform isoamyl alcohol extracting:
Through step c) viral supernatant after treatment precipitate with PEG, the resuspended precipitation of buffer solution, viral resuspended liquid is through taking out containing extractCarry, described extract obtains after chloroform and isoamyl alcohol are mixed by 24:1 volume ratio;
The quality control index of this step is: foot and mouth disease virus titre is 107.0-108.0logTCID50/ mL, total protein concentration is 3-4.2 grams, foot and mouth disease virus 146S content is 50-60 μ g/ml, nonstructural protein 3A BC residual volume≤15ng/ml, bovine serum albuminWhite residual volume≤90ng/ml, BHK-21 cell rests protein content≤60ng/ml, BHK-21 cell rests DNA amount≤3500pg/Ml, Triton-X-100 residual volume≤400 μ g/ml, PEG residual volume≤250 μ g/ml, chloroform residual volume≤350 μ g/ml,Endotoxin≤55EU/ milliliter;
E) deactivation;
0.025% divinyl imines, 4 DEG C of deactivations 48 hours or 37 DEG C of deactivations 2 hours;
F) SDGC purifying:
Step deactivation liquid e) is by Continuous Flow 60% sucrose isodensity gradient ultracentrifugation purifying, this step quality control indexFor: total protein concentration 2-2.5 gram, foot and mouth disease virus 146S content is 20-30 μ g/ml, nonstructural protein 3A BC residual volume≤4ng/Ml, bovine serum albumin(BSA) residual volume≤50ng/ml, BHK-21 cell rests protein content≤60ng/ml, BHK-21 cell restsDNA amount≤2000pg/ml, Triton-X-100 residual volume≤250 μ g/ml, PEG residual volume≤200 μ g/ml, chloroform is residualSurplus≤400 μ g/ml, endotoxin≤800EU/ milliliter;
G) ultrafiltration dialysis, aseptic filtration:
Through step f) virus liquid after purifying through ultrafiltration dialysis concentrated 25-50 doubly, virus liquid, should through 0.22 μ m film aseptic filtrationStep quality control index is: total protein concentration 2-2.5 gram, foot and mouth disease virus 146S content is 20-30 μ g/ml, non-structural protein3ABC residual volume≤3ng/ml, bovine serum albumin(BSA) residual volume≤50ng/ml, BHK-21 cell rests protein content≤30ng/ml,BHK-21 cell rests DNA amount≤100pg/ml, chloroform residual quantity≤20 μ g/ml, PEG residual volume≤10 μ g/ml,TritonX-100 residual volume≤15 μ g/ml, endotoxin≤35EU/ milliliter;
H) stoste deposit or emulsification preparation.
2. the method for claim 1, is characterized in that in the stepwise elution described in step c) that washing lotion is for containing 90mMThe 4.7mM sodium phosphate buffer of the pH6.5-7.0 of sodium chloride, with pH7.5 containing the sodium phosphate of the 120mM of 0.35M sodium chloride andThe buffer solution stepwise elution of 1mMEDTA, every 1000 cubic centimetres of ion-exchanges absorption bed or film are processed the virus liquid of 40-50ml.
3. the method for claim 1, is characterized in that steps d) described in PEG be PEG-8000 or PEG-6000,Virus liquid through nuclease digestion, strong anion exchange absorbing bed purifying wash-out precipitates with PEG, makes dense in virus liquid of PEGDegree 2.7-10% (W/V), sodium chloride concentration is 0.35-1M, 2-8 DEG C of vigorous stirring 1 hour, preparative centrifuge with 1000g fromThe heart 10 minutes, removes supernatant, with containing 120mM sodium chloride, 6.2mM sodium phosphate, the resuspended precipitation of 1mMEDTA (pH7.5) buffer solution, bodyAmass the 10-20% for virus-culturing fluid.
4. the method for claim 1, is characterized in that during step f) that deactivation liquid is by Continuous Flow 60% sucrose isodensityGradient ultracentrifugation purifying, centrifuge used is manufacture type series, and large capacity rotary head volume is 3.2-8 liter, and batch processing is taken out2000 liters of extracts; Foot and mouth disease virus deactivation liquid adopts density gradient ultracentrifugation, and ultracentrifugation mode is Continuous Flow, and gradient is not forContinuous gradient, comprises the gradient that (1) centrifugal force 36000-60000g forms; (2) ladder that centrifugal force 90000-120000g formsDegree; (3) the flow velocity 10L/ hour of foot and mouth disease virus deactivation liquid; Level pad used is the 0.04MPBS of pH7.2-7.6,60% sucrose solution adopts the 100mMNaCl that contains of pH7.6, and the 0.04M phosphate buffer of 0.1%Triton-X-100 is joinedSystem, rotor injects 1.6 liters of 60% sucrose solutions that prepare; With 20 ls/h of loadings, process foot and mouth disease virus PEG precipitation resuspended2000 liters of liquid, rotating speed 32000rpm.
5. the method for claim 1, is characterized in that strong anion exchange absorbing bed or the film described in step c) isRefer to QSepharoseXL ion-exchange absorption bed or film.
6. the method for claim 1, is characterized in that the adjuvant that the emulsification described in step h) adopts is V201.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410643314.3A CN104491855B (en) | 2014-11-07 | 2014-11-07 | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410643314.3A CN104491855B (en) | 2014-11-07 | 2014-11-07 | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104491855A CN104491855A (en) | 2015-04-08 |
| CN104491855B true CN104491855B (en) | 2016-05-25 |
Family
ID=52933368
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410643314.3A Active CN104491855B (en) | 2014-11-07 | 2014-11-07 | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104491855B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845947A (en) * | 2015-04-18 | 2015-08-19 | 湖北创瑞生物科技有限公司 | Purification method of recombinant herpes simplex virus |
| CN104892734B (en) * | 2015-04-30 | 2019-09-13 | 中国科学院过程工程研究所 | The method of hydrophobic interaction chromatography purification aftosa inactivation of viruses antigen |
| CN104818254A (en) * | 2015-05-06 | 2015-08-05 | 中国科学院过程工程研究所 | Method of purifying foot-and-mouth disease inactivated virus antigen through ion exchange chromatography |
| CN104826097A (en) * | 2015-05-11 | 2015-08-12 | 北京健翔和牧生物科技有限公司 | Preparation method of foot-and-mouth disease vaccines |
| CN107064488B (en) * | 2017-05-02 | 2019-04-16 | 中国农业科学院兰州兽医研究所 | A kind of total antibody solid phase of swine fever virus serum blocks the preparation method of antigen used in ELISA kit |
| CN109535230A (en) * | 2017-09-21 | 2019-03-29 | 中国科学院过程工程研究所 | The method for preparing foot and mouth disease virus vaccine antigen based on metal chelate affinity chromatography |
| CN108531462B (en) * | 2018-04-20 | 2021-03-30 | 华南农业大学 | Method for purifying avian adenovirus essence |
| CN108676780B (en) * | 2018-07-24 | 2021-08-10 | 华中农业大学 | Method for efficiently separating porcine enterocoronavirus |
| CN109134622B (en) * | 2018-08-16 | 2021-07-02 | 金宇保灵生物药品有限公司 | Multistep continuous integrated purification method for foot-and-mouth disease virus antigen |
| CN114230641B (en) * | 2021-12-29 | 2024-03-26 | 内蒙古必威安泰生物科技有限公司 | Method for removing endotoxin in foot-and-mouth disease antigen liquid |
| CN115873810B (en) * | 2022-12-26 | 2024-02-09 | 苏州良辰生物医药科技有限公司 | Purification method of murine leukemia virus |
| CN116200244B (en) * | 2023-05-04 | 2023-09-15 | 赛奥斯博生物科技(北京)有限公司 | Separation and purification equipment and purification method for cell culture virus liquid |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130072A (en) * | 2007-07-23 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Type A inactivated vaccine for hoof-and-mouth disease |
| CN103087995A (en) * | 2012-12-28 | 2013-05-08 | 杭州国牧生物科技有限公司 | Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells |
| CN105126096A (en) * | 2015-09-08 | 2015-12-09 | 吕宏亮 | Foot and mouth disease virus-like particle vaccine and preparation method thereof |
-
2014
- 2014-11-07 CN CN201410643314.3A patent/CN104491855B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101130072A (en) * | 2007-07-23 | 2008-02-27 | 中国农业科学院兰州兽医研究所 | Type A inactivated vaccine for hoof-and-mouth disease |
| CN103087995A (en) * | 2012-12-28 | 2013-05-08 | 杭州国牧生物科技有限公司 | Method of preparing foot-and-mouth disease vaccine by using BHK-21 adherent cells |
| CN105126096A (en) * | 2015-09-08 | 2015-12-09 | 吕宏亮 | Foot and mouth disease virus-like particle vaccine and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 口蹄疫疫苗生产质量控制技术的现状与未来;谢文强;《广东畜牧兽医科技》;20121231;第37卷(第3期);第6、8页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104491855A (en) | 2015-04-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104491855B (en) | Method of the aftosa whole virus particles marker vaccine of a kind of extensive preparation high yield, high-purity, high safety and products thereof | |
| US11013794B2 (en) | Method for preparing foot-and-mouth disease vaccines | |
| CN107267466B (en) | Method for large-scale production of porcine pseudorabies inactivated vaccine | |
| CA2197683C (en) | Method for preparing an influenza virus, antigens obtained and applications thereof | |
| CN103374547B (en) | A kind of preparation method of foot and mouth disease purified vaccine | |
| US6149917A (en) | Industrial production process for a vaccine against Japanese encephalitis virus and vaccine produced | |
| CN104826101B (en) | Human lyophilized rabies vaccine and preparation method thereof | |
| CN111920944B (en) | Preparation method of influenza virus subunit vaccine stock solution | |
| CN107384877A (en) | A kind of purification process of slow virus | |
| CN107567496A (en) | Axenic purification method about virus | |
| CN107254449A (en) | A kind of method for mass producing high-purity PRV | |
| CN106540249A (en) | A kind of bird flu (H5N1) or the antigen concentrating and purifying process of Porcine reproductive and respiratory syndrome (PRRS) viral vaccine | |
| CN101020053B (en) | Preparation process of inactivated rotavirus vaccine | |
| CN102133399B (en) | Novel process for preparing influenza virus split vaccine | |
| CN105349499A (en) | Preparation method, product and application of avian influenza whole-virus particle marked vaccine | |
| CN106754765A (en) | A kind of NDV sample particle, preparation method and applications | |
| CN103937754B (en) | Porcine reproductive and respiratory syndrome virus (PPRSV) purification method | |
| CN109395074A (en) | A kind of encephalitis B inactivated vaccine lyophilized preparation and preparation method thereof | |
| CN104548085B (en) | A kind of aftosa whole virus particles vaccine combination and its preparation method and application | |
| CN102533679B (en) | Preparation of virus release buffer solution and application thereof | |
| Ray et al. | Glycoproteins of human parainfluenza virus type 3: affinity purification, antigenic characterization and reconstitution into lipid vesicles | |
| CN106279376A (en) | A kind of pig circular ring virus antigen purification method | |
| CN116162601A (en) | Preparation method of influenza virus split vaccine | |
| CN103768590A (en) | Method for removing residual DNA in encephalitis B vaccine product by utilizing anionic exchange chromatography | |
| CN116966287B (en) | Cracking method for influenza virus cracked vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |