CN100398642C - Improvements in virus production - Google Patents

Abstract

An improved process for recovery of virus from allantoic fluid of virus-infected chick embryos. Virus associated with granular and fibrous debris in the allantoic fluid can be disassociated from the debris and recovered, thereby increasing viral yield. Dissociation can be achieved by subjecting the virus-debris complex to conditions of increased salt concentrations, e.g., 0.5 M or greater.

Description

The improvement of virus production

The application requires the U.S. Provisional Application sequence number 60/429 of application on June 20th, 2003,723, the U.S. Provisional Application sequence number 60/540 of application on January 30th, 2004, the U.S. Provisional Application sequence number 60/572 of application on May 20th, 782 and 2004,718 right of priority, all these merge to its integral body among the present invention by reference at this.

Invention field

The present invention relates to from the allantoic fluid of the chicken embryo of virus infection, reclaim enveloped virus.The rate of recovery that improves helps to produce virus vaccines, particularly influenza vaccines, and the virus protein of high yield also can be provided, comprising the foreign protein matter of being expressed by virus vector.

Background of invention

In case by pathogenic infection, host's immunity system just can identify on pathogenic agent or the intravital antigen of cause of disease, and the antagonism of mediation immunne response contains antigenic pathogenic agent.In this answering, the immunocyte quantity of the antigen-specific of pathogenic agent is increased, and some the still existence infection is calmed down after in these cells.The cell of these existence stops pathogenic agent to set up infection when the host is subjected to these pathogenic agent invasion and attack afterwards.This is called protective immunity.

Under the situation that does not cause disease, vaccine is by presenting to the protective immunity that immunity system provides the enantiopathy substance with the antigen of pathogenic agent.Developed certain methods, do not caused in pathogenic agent to allow antigen to exist under the situation of disease infection.These methods comprise the fragment (subunit) of using attenuated pathogens, inactivating pathogens or the pathogenic agent of living.

Because to the treatment of many virus infectiones unpredictable still, therefore preferably prevent or alleviate infection by vaccine inoculation, rather than infect occur after the treatment infection.The example of thorny especially infective virus is those orthomyxoviridae family (orthomyxoviridae), particularly influenza virus, Paramyxoviridae, flaviviridae, batch Tectiviridae, Rhabdoviridae and coronaviridae.Millions of people's preventive vaccination is arranged to resist in these virus families one or more every year.

Although some virus is bred finely in cell culture, virus need be bred and be reclaimed to other viruses from allantoic fluid in containing the egg of embryo.For many years, influenza vaccines offer masses as the joint product of the many bacterial strains that reclaim from the allantoic fluid of the egg that contains embryo.From large quantities of bacterial strain samples, select every year three kinds of bacterial strains to cultivate, purifying, and collect to produce specified vaccine.The growth of each selecteed influenza bacterial strain can change significantly, thereby usually causes being difficult to satisfying effectively the annual market requirement to a kind of like this trivalent vaccine.

The whole bag of tricks has been proposed to improve and/or to simplify and from raw material, reclaim virus or viral product.US 3,627, and 873 disclose and use ether and methyl acetate to extract viral method from spissated allantoic fluid raw material.According to US 4,000,257 reports use butylacetates and ethyl acetate to carry out the multiple extraction productive rate that further improves of having obtained.

US 3,316, and 153 disclose a kind of multistep extracting method, and this method is intended to separating viral particles from cell debris, and declare this method to be used for from the chicken allantoic fluid of virus infection or the raw material of cell or tissue nutrient solution acquisition.In the method, the virus that is adsorbed on the calcium phosphate precipitation is dispersed in the EDTA under pH7.8-8.3, cause to dissociate and EDTA chelating soluble calcium, thereby releasing virus is for recovery.Resulting solution with water or the preferred glycine-sodium chloride aqueous solution that contains virus dialysed to reduce EDTA and phosphate content.

U.S. Patent No. 4,724,210 disclose the method for using the ion-exchange chromatogram purification influenza virus.For example allantoic fluid is by the sulfate cellulose post for the solution that will contain influenza virus, and virus is adsorbed on the weighting material of post in this post.With this pillar of after scouring, and with the sodium chloride solution wash-out virus that contains 1.0M-1.5M.Then the sodium-chlor with 4.99M washs.

In WO 02/067983, the preparation method of cracking influenza virus vaccine is disclosed, comprising middling speed centrifugal purification allantoic fluid, the liquid adsorption that makes purification is to CaHPO ₄On the gel, then use EDTA-Na ₂Solution dissolves again.Same referring to the WO 02/08749 that discloses same procedure.

At US 4,327, in 182, the allantoic fluid raw material experience multistep extraction process of growth influenza virus, this extraction process is intended to reclaim influenza virus subunit, hemagglutinin (HA) and neuraminidase (NA).This technology depends on enrichment step, in this step viral raw material and stain remover is in the same place with salt solution mix, follows by continuous filtration to remove non-virion.

U.S. Patent No. 3,962,421 disclose the method for cracking influenza virus.Allantoic fluid is carried out high speed centrifugation.Resulting precipitation be resuspended in the physiological saline and ball milling 12-15 hour to obtain viral suspension.Handle this viral suspension with phosphoric acid ester then, virion is cracked into the particle that does not contain liposome (subunit) that carries the intact virus surface antigen.

At US 3,874, in 999, low-speed centrifugal contains the allantoic fluid of influenza virus to remove coarse particles.Isolating virus by high speed centrifugation from supernatant liquor then also should virus be suspended in the phosphate buffered saline buffer again.Under the alkaline pH condition, under 4 ℃, handle suspension 16-18 hour to remove non-virus protein and liposome with the sal epsom of 0.1-0.4M.Low-speed centrifugal purifies the gained suspension, and from the gained supernatant liquor purified virus.

To the interested especially research that is the recycling of virus and various salt concn to the influence of the virus that is purified in the background technology of the present invention, comprising non-allantoic fluid viral source is contacted with the solution of one or more salt with rising concentration.

Certain methods is declared can provide the virus or the highly purified virus of high yield when infected cells and the solution that contains high salt concentration being contacted or cultivating subsequently from this solution purified virus.

In WO 99/07834, will in the salt brine solution (for example 0.8-0.9M NaCl) that height oozes, be cultivated a few hours by the Vero cell culture of herpesvirus infection.Remove solution and the simplexvirus from collect solution then.This method is declared to be better than cell is carried out the method for ultrasonic degradation.

Other method has related to making by the culturing cell of virus infection and has contacted with high salt concentration.

US 5,506,129 reported infected BS-C-1 cell cultures is being contained～developing medium of 0.3M NaCl in after, the productive rate of hepatitis a virus improves.

Resulting rabies virus lacks the oversensitive activity that is caused by residual cerebral tissue Karakuyumchan etc. (Acta virol.155-158,1981) have reported the infected cerebral tissue that vibrates in the buffered soln that contains 0.3M NaCl after.

Pauli and Ludwig (Virus Research, 2:29-33,1985) have reported that will infect viral clone cultivates and containing～obtaining in the medium of 0.3M NaCl the borna disease virus (Borna disease virus) of high yield.

Different study group has studied the virus that is purified and has contacted influence to virus characteristic with the salt concn of rising.

In (Virology, 80:441-444,1977) such as Breschkin, the Measles virus that lacks the active specific sudden change of erythrocyte agglutination in isotonic saline solution is at 0.8M (NH ₄) ₂SO ₄In have the erythrocyte agglutination activity of wild-type level, and hypermutation is to the active not influence of the erythrocyte agglutination of the virus of wild-type.

Wallis and Melnick (Virology, 16:504-506,1962) report: although high salt (1M MgCl ₂, 1M CaCl ₂, or 2M NaCl) can prevent the heat inactivation of poliovirus, Coxsackie virus and echo, enteric cytopathogenic human orphan virus, but 1M MgCl ₂Can strengthen the inactivation of adenovirus, many empty (papova) viruses of breast, simplexvirus, myxovirus, arbor virus and poxvirus.

In (Acta virol.27:407-411,1983) such as Willkommen, the freeze-drying influenza virus of purifying is restored in containing the buffer saline that rises NaCl with high concentration (at most to 1.15M).Subsequently, should and carry out one-way radiation shape immunodiffusion(ID) (SRD) test by restorative virus with the stain remover cracking.Under the situation of the bacterial strain that adopts some influenza viruses, in the restorative damping fluid, increase salt concn and show that the SRD test-results to hemagglutinin (HA) does not influence.Yet when other bacterial strain is containing when being restored in the buffer saline of 1.15M NaCl, the HA concentration that they provide in the SRD test is 2 times that identical bacterial strain is containing HA concentration when being restored in the buffer saline of 0.15M NaCl.The author confirms that viral gathering will block the infiltration of stain remover as far as possible and weakened SRD and replied.

Molodkina etc. (Colloids and Surfaces A:Physicochemical and EngineeringAspects, 98:1-9,1995) have reported that salt concn is increased to 0.3M NaCl can make the influenza virus aggregate of purifying disperse.

Sudnik etc. (Vyestsi Akademii Navuk BSSR Syeryya Biyalahichnykh Navuk, 6:71-77,1985) have reported that under pH2.2 high ionization degree energy partial offset is to the destruction of influenza virus coating.

Interested in the background technology of the present invention also have (Voprosy Virusologii, 34 (2): 274-279) about the result of study of thread influenza virus body ratio in the allantoic fluid such as Makhov.In the bacterial context of separating in virus reclaims, Makhov etc. have reported that the existence of thread influenza virus body is that bacterial strain is special and relevant with ionic strength in the allantoic fluid.Applying electronic microscopic examination allantoic fluid is to measure existing of inovirus body.For a kind of specific influenza virus bacterial strain, the occurrence rate of inovirus body is 7.1% in allantoic fluid.When NaCl concentration was elevated to 0.25M or 1.0M, occurrence rate was reduced to 0.37% and 0.16% respectively.

Therefore, this area still needs to improve purity and the productive rate that obtains virus from the allantoic fluid of the chicken embryo of virus infection.

Summary of the invention

The invention provides and from the allantoic fluid of the chicken embryo of virus infection, reclaim improving one's methods of virus.Just as disclosed in this, have now found that the virus that considerable part is arranged combines with granular or fibrous fragment in allantoic fluid, therefore when the purification allantoic fluid was removed fragment, they were lost thereupon.Before final separating treatment,, virus improves viral productive rate from fragment by being separated.

The processing method that reclaims virus from allantoic fluid usually comprises the step that allantoic fluid is purified, for example by centrifugal or filter to form the liquid ingredient that purifies and to contain the component of fragment.When by the centrifugal purification allantoic fluid, the component that contains fragment typically is precipitation (pellet) form; When by the filtration, purification allantoic fluid, the component that contains fragment typically is trapped substance (retentate) form.By comprising virus is contained this step that extracts the component of fragment from this, the invention provides the known method that from the allantoic fluid of the chicken embryo that infects, reclaims virus of improving.

The preferred method that extracts virus from the allantoic fluid component that contains fragment is that virus is dissociated in the suspension, and this suspension has the non-isotonic concentration of one or more salt.Especially, adopt the solution that contains one or more salt, total salt concn is about the salt/ml allantoic fluid of 0.5M or bigger or a great deal of mol ratio in described this salts solution, and virus can easily disintegrate down from fragment.Useful especially solution is pH scope from 3 to 10 and the phosphate buffer soln that comprises the total salt concentration (for example total NaCl concentration) from about 1.0M to about 3.5M.Reclaimed from this suspension by dissociated virus.Recovery comprises that purification forms second kind of decontaminating liquid and second kind of component that contains fragment for the second time.The preferred method that reclaims virus also is included in location virus on the sucrose density gradient.

In certain aspects, the present invention makes the total salt concentration in the solution be about 0.5M or bigger by one or more salt are joined in the allantoic fluid, reclaims virus subsequently from gained liquid the method that reclaims virus from the allantoic fluid of the chicken embryo of virus infection is provided.Salt can be the form adding with the aqueous solution, as strong phosphoric acid salt buffer (PBS).In one embodiment, before allantoic fluid is separated from egg, salt is joined in the allantoic fluid.After adding one or more salt, the recovery of virus comprises that a purifying step contains the component of fragment with formation.Extraction remains in any virus in the component that contains fragment, with the productive rate optimization of virus.Perhaps, removing or do not having except that anhydrating with under reducing by the situation of fractionated liquid volume, the allantoic fluid that salt was handled directly carries out processing treatment with for example sucrose density gradient classification.

In an especially preferred embodiment, the solution that will contain one or more salt join in the allantoic fluid so that in the solution total volume concentration be about 1.5M or bigger.Also can regulate or keep the pH of allantoic fluid.The preferable range of pH comprises pH3.0-pH6.8 or pH6.8-pH9.8.After joining salt in the allantoic fluid, make it to purify by centrifugal or filtration.Then the allantoic fluid that purifies being carried out sucrose density gradient separates to locate virus.Subsequently, from this gradient, localized virus is separated.

Use method of the present invention can be recovered in any virus of duplicating and in allantoic fluid, existing in the chicken embryo of virus infection basically.Particularly preferred virus is the coating RNA viruses, comprising the member of orthomyxoviridae family, Paramyxoviridae, flaviviridae, batch Tectiviridae, Rhabdoviridae and coronaviridae family.As what confirmed among the embodiment, method of the present invention has significantly improved the two the rate of recovery of influenza virus A and influenza virus B.

Other features and advantages of the present invention may be obvious that from the following detailed description.Yet, be to be understood that, although the description that this is detailed and specific embodiment expression the preferred embodiments of the invention, but only the mode by explanation provides, because according to this detailed description, various changes within the spirit and scope of the present invention and modification all are conspicuous concerning skilled those skilled in the art.

Description of drawings

Fig. 1: the precipitation of getting off from the allantoic fluid that purifies with the solution-treated that contains 1.6M NaCl has improved productive rate and better location is provided in saccharose gradient.

The description of preferred embodiment

On the one hand, the invention provides and from the allantoic fluid of the chicken embryo of virus infection, reclaim improving one's methods of virus.This method has significantly improved the productive rate that reclaims virus from allantoic fluid, and the highly purified viral composition that can be used for preparing vaccine is provided, or deutero-viral sub-units goods.

As herein and employed in the claims, " virus " is meant the virion of coating, and preferred complete sum has communicable, rather than viral fragment, component and/or independent virus antigen, for example obtains by known cracking technique.Reclaim after the virus according to the present invention, the fracture of virion or cracking are very easy to.

Method of the present invention can be used for the virus of naturally occurring virus and genetic modification, for example those described in WO 99/66045 and its corresponding communique US 2003/0087417.

In a preferred embodiment, the chick embryo allantoic liquid for preparing virus infection according to the guide that is used for production of vaccine of present foundation.Usually, what this method need be used the 9-12 age in days contains the embryo egg, and these eggs are addled with rejecting by illumination (precandled) or the egg of unfertilized (unfertilized) in advance.To wish to obtain the specific live virus inoculation of vaccine then in the amnion and/or allantoic cavity of all the other eggs.Generally under 32-37 ℃, egg was hatched 2 or 3 days, shine the egg that egg (post-candled) addles with rejecting afterwards, then under about 4-6 ℃ temperature, these cold storage of eggs were then gathered in the crops egg liquid in about 24 hours under sterile state.So the allantoic fluid of results contains the live virus of high density.This method especially can be used for producing various types of influenza viruses, comprising most or all bacterial strains of influenza virus A and influenza virus B.

As what confirmed among the following embodiment 1, although the allantoic fluid of virus infection contains the live virus of high density, but many viruses combine (gathering) together with fibrous or granular fragment, and when fragment was separated from allantoic fluid basically, these viruses were lost thereupon.By using the salt concn that raises to make virus disintegrate down from fragment and reclaim by dissociated virus, method of the present invention has improved the productive rate that reclaims virus from allantoic fluid.Before any purification, (after perhaps separating from egg in the egg that infects or with allantoic fluid) can disintegrate down virus from fragment in allantoic fluid.In fact, in some instances, before for example sucrose density gradient is separated, can remove purification from as preliminary recycling step.Perhaps, allantoic fluid purify can be formed the component that contain fragment, from this contains the component of fragment, virus be dissociated out from fragment subsequently.After fragment disintegrates down, use the viral purification technology of routine as described below to reclaim virus in virus.

The preferred method that from the accumulative fragment virus is disintegrated down is to have non-grade and ooze in the environment of salt concn being placed at fragment bonded virus.It is reported that when it significantly is different from the salt concn of allantoic fluid environment has " non-grade is oozed " salt concn, the total salt concentration of described allantoic fluid is about 150mM.The example that non-grade is oozed salt concn includes, but not limited to 10mM or still less, 20mM or still less, 30mM or still less, 40mM or still less, 50mM or still less, 60mM or still less, 70mM or still less, 80mM or still less, 90mM or still less, 100mM or still less, 110mM or still less, 120mM or still less, 130mM or still less, 140mM or still less, these concentration can obtain by the dilute with water allantoic fluid.Ooze salts solution such as phosphate buffered saline buffer dilution and can not make allantoic fluid become non-etc. oozing with waiting, but as described in following, just dissociating from fragment virally can have useful effect.

Non-grade is oozed salt concn and is comprised that height oozes salt concn, 160mM or bigger for example, 170mM or bigger, 180mM or bigger, 190mM or bigger, 0.2M or bigger, 0.3M or bigger, 0.4M or bigger, 0.5M or bigger, 0.6M or bigger, 0.7M or bigger, 0.8M or bigger, 0.9M or bigger, 1.0M or bigger, 1.1M or bigger, 1.2M or bigger, 1.3M or bigger, 1.4M or bigger, 1.5M or bigger, 1.6M or bigger, 1.7M or bigger, 1.8M or bigger, 1.9M or bigger, 2.0M or bigger, 2.5M it is or bigger, 3.0M or bigger and 3.5M or bigger, and can be by direct adding free salt or preferably obtain by adding dense saline solution.

In all embodiments, one or more salt are joined realized in the allantoic fluid virus is disintegrated down from the accumulative fragment.Dissociate in case virus takes place, then before reclaiming virus, can dilute the solution that contains virus once more, for example make this solution becomes to wait and ooze (being that the high degree of oozing reduces).Perhaps, before adding salt or simultaneously, the dilution allantoic fluid afterwards can be with the solution concentration of dilution improving salt concn, thereby before reclaiming virus, virus is dissociated out from the accumulative fragment.In these embodiments, obtained the preferred molar ratio of the original volume of salt and allantoic fluid.

For example, in the example 10 below, the allantoic fluid by the 1 * PBS that adds 50ml dilutes the 100ml five equilibrium makes sample volume become 150ml.20 * PBS with equal-volume (150ml) joins in this sample subsequently, obtains the final volume of 300ml.Allantoic fluid and 1 * PBS have the NaCl concentration of about 0.15M (150mM).Therefore, 0.015mol NaCl (0.1L * 150mM NaCl) is arranged in initial allantoic fluid.Use 1 * PBS dilution then, and interpolation 0.0075molNaCl (0.05L * 150mMNaCl).20 * the PBS that is added increases 0.45mol NaCl (0.15L * 3.0M NaCl).The final volume of 300ml contains the NaCl (0.015+0.0075+0.45) of 0.4725mol.Therefore, the mol ratio of salt and allantoic fluid is 0.4725mol/100ml allantoic fluid or 4.725mmol/ml allantoic fluid raw material.If allantoic fluid is adjusted to 0.5MNaCl, then mol ratio is a 1.5mmol NaCl/ml allantoic fluid raw material, and with (always) volume-independent that is conditioned.

Preferred salt is those salt that are regarded as safety (GRAS) that use in the human medicine usually.Preferred salt is sodium-chlor.This salt also can be prepared from and can be comprised or get rid of particularly ammonium sulfate by its unit price, divalence or polyvalent cation mixture.Therefore, KCl, LiCl, CaCl ₂, MgCl ₂Be regarded as making up salt with other salt.Other change comprises various inorganic salt and organic salt (for example, sodium acetate, potassium acetate etc.).In the embodiment of using high salt concentration that virus is dissociated out from the accumulative fragment, selected in the methods of the invention salt should be that those are satisfying the soluble salt of maintenance under the high density of required environmental requirement.Any degree of ionization (penetration degree) that can significantly improve solution keeps the salt of solubleness all to be suitable for again simultaneously.This salt comprises sodium-chlor and Repone K and analogue.

In a preferred embodiment, will be with the allantoic fluid and the aqueous solution that contains high salt concentration of the virus infection of known manner preparation, so that the volumetric molar concentration of the salt that the gained mixture contains is 0.5M and saturated at most at least, better volumetric molar concentration is in the 1.0M-3.5M scope.Usually this can realize, for example by isopyknic allantoic fluid of mixing and salts solution, or uses any other mixed method that required salt concn can be provided.In some embodiments, before adding salts solution, allantoic fluid is separated from egg.In other embodiments, salts solution is joined in the allantoic fluid in the egg, or after having collected most of allantoic fluids, use the washed with saline solution allantoic cavity.

Preferably, the mixture of allantoic fluid and salt also can be cushioned, and for example uses phosphate buffer (for example 20-250mM) to obtain required pH by general method.Preferred pH scope is from 3.0 to 10.0.Based on viral one by one, can regulate pH and make rate of recovery maximum.Be fine-tuning to for further improving productive rate in given Virus Type or the hypotype preferred range by pH dense salt/raw mix.Preferred pH scope is from 3.0 to 10.0.For example, when non-grade ooze environment have in the 6.8-7.1 scope neutral relatively/slightly during acid pH, the productive rate of the Moscow bacterial strain of influenza virus A is higher.Yet under the condition of identical salt, about 8.4 when non-grade is oozed environment and had higher pH level, the productive rate of some Yamanashi bacterial strains of influenza virus B is bigger.

Virus can further be comprised divalent cation chelators from the environment that fragment disintegrates down, as EDTA.Sequestrant plays the effect of the purpose of chelating divalent cation, wherein divalent cation influences the precipitation or the gathering of virion, thereby make titer determination not detect them, perhaps make them can not touch employed deactivation condition in the technology in production of vaccine stage.The concentration of suitable sequestrant is to impel isolating those concentration of accumulative virion.Under the situation of using EDTA, concentration is adapted in the 1.0mM-1.0M scope, and this depends on the concentration of initial divalent cation.Other additive can be separately or combination, comprises reductive agent, as DTT, and wetting agent, as nonionic detergent Triton X-100 and Pluronic F68.

As mentioned above, in some embodiments, one or more salt are joined in the allantoic fluid of chicken embryo of virus infection to improve the salt concn in it.In some embodiments, need the mixture of allantoic fluid and dense saline solution, wherein for convenience's sake, at room temperature (18-25 ℃), described mixture pH can randomly be regulated and be contained optional sequestrant.This mixture can be stirred.Do not have under the sedimentary situation this mixture to be cooled off to keep viral infectivity at salt then, this contains viral suspension is the remaining period of stirring not having, and perhaps passes through centrifugal or filtration formation ideally.

Then optionally processing treatment this contain virus supernatant liquor or filtrate with further enrichment virus.Typically, by handling the known mode of thick allantoic fluid, then supernatant liquor is carried out sucrose gradient centrifugation and handle, this processing is positioned at virus on this gradient or in the gradient.From this gradient, reclaim the virus that is positioned, obtain containing the component of virus.After sepn process, obtained the component that contains virus of one or more merging, formed the extract that is rich in virus based on sucrose.This extract that is rich in virus also can carry out hypermutation to be handled, thereby prevents or remove the viral aggregate that is caused by the gradient purifying usually.

Be to be understood that, method of the present invention is not limited to any specific classification or separation method, can adopt on the contrary any other to from allantoic fluid, obtaining useful classification or the separation method of purified virus, comprising those methods and the similar approach of using size exclusion chromatography, centrifugal, filtration, solvent extraction, ion-exchange chromatography.In addition, can make any one or a plurality of obtained component is non-etc. oozing, to improve the recovery technology of virus, because virus singly is dispersed in the solution basically.

In some embodiments, the virus of recovery is inactivated.The method of deactivation can be known any method in those in production of vaccine, comprising formalin fixed, radiation, stain remover or solvent cracking and similar approach.

The present invention can be used for reclaiming the virus with the purifying wide region.In preferred embodiments, this method is used to reclaim the enveloped virus that contains rna gene.This viroid comprises those in orthomyxoviridae family (for example influenza virus), Paramyxoviridae (for example mumps virus, Sendai virus and Avian pneumo-encephalitis virus), flaviviridae (for example japanese encephalitis virus and yellow fever virus), batch Tectiviridae (for example rubella), Rhabdoviridae (for example vesicular stomatitis virus and rabies virus) and coronaviridae (for example bird the infects bronchitis virus) family.In specific embodiment, this method is used to reclaim influenza virus, comprising the bacterial strain of influenza virus A, influenza virus B, influenza virus C, avian influenza viruses, horse parainfluenza virus and Swine influenza virus.

As mentioned above, in practices more of the present invention, before reclaiming virus, the dilution allantoic fluid.In others, with purifying the component resuspending that contains fragment that obtains at first, handle so that virus discharges from fragment with high salt, purify again, and the solution of resulting this purification is returned in the solution that is added to initial purification.Therefore, the volume of solution that contains virus in the course of processing can increase.

The solution that contains virus of handling large volume may be loaded down with trivial details, especially when reclaiming virus from sucrose density gradient.Do not having under the situation of significantly loss virus, the method that reduces the volume of the solution that contains virus is known in this area.For example, the solution that contains virus can be cut and flow through filter (TFF) or diafiltration (diafiltration).In TFF, the virus flows in the solution is crossed the tubular fibre strainer tube or passed the filter material flitch.With feedstream to be in the normal flow filtration of equidirectional opposite with pressure, TFF depends on and the vertical pressure of raw material flow direction.Therefore, in TFF, filtrate flow is through the membranous wall that contains of chimney filter, and trapped substance flows downward along the path of strainer tube simultaneously.In this technological process, can reduce liquor capacity on demand.

In some embodiments, hope will contain virus solution carry out diafiltration.In the diafiltration process, the tensio-active agent, protein or other solute that freely see through filtering membrane are removed from solution.In general, two kinds of diafiltration patterns commonly used are arranged: intermittence and constant volume.In intermittence diafiltration process, add the damping fluid or the solution of large volume, concentrate trapped substance then.In the diafiltration process of constant volume, add damping fluid or solution to flow out identical speed with filtrate.

The solution that contains virus is carried out the film that uses in TFF or the diafiltration commercially available (MILLIPORE for example, Billerica, MA).In preferred embodiments, the scope of holding back of film is the 100kD-0.05 micron.

In some embodiments, the present invention is used for purifying by one or more protein of encoding viral and in some cases, is secreted into protein in the allantoic fluid by the protoblast that infects.Protein can be virus protein or by the alien gene encoded protein matter that is included in the recombinant viral vector.In preferred embodiments, one or more salt are joined contain in this proteinic allantoic fluid so that protein disintegrates down from fragment.Perhaps, will with fibre debris bonded protein for example by centrifugal or filter and from allantoic fluid, to separate, the component that will contain fragment then places non-grade to ooze salt concn to make protein dissociate out from fragment.The protein that uses the standard technique purifying to be disintegrated down subsequently.

When the present invention being applied to from the chicken allantoic fluid when reclaiming influenza virus, egg quantity of the presently claimed invention significantly descends for a specified influenza vaccines production cycle.This has reduced the possibility of vaccine shortage when annual influenza begins season again.Other advantage also comprises the handling problem that is easy to of amount of work minimizing and refuse.All these advantages have significantly reduced the cost of producing influenza vaccines.

Be used for from the specific embodiments of the present invention of allantoic fluid extraction influenza virus, can using method of the present invention with following ad hoc fashion.

Results contain the infected allantoic fluid of high titre influenza virus vaccine bacterial strain under the industrial procedure of standard, and handle immediately or before further handling, (for example under-70 ℃) store under lyophilised state.

When adding man-hour, NaCl with the 16-20% (w/v) of isopyknic phosphate buffered handles liquid allantoic fluid, usually in the scope of 6.5-8.5, this depends on influenza virus bacterial strain to be purified to pH, and can contain or not contain sequestrant such as EDTA or other additive.After suitably cultivating, about 5 minutes or longer,, purifying in the viruses from solution is come out by sucrose density is centrifugal when from fragment, disintegrating down at the allantoic fluid inner virus.Perhaps, can use a step or a multistep purifying step.In some embodiments, by at maximum 14000 * g, for example purify virus product centrifugal time of 2000-5000 * g.Compare with the supernatant liquor that is not subjected to high salt processing, this supernatant liquor significantly is rich in live virus.Sedimentary fragment can be randomly with the solution-treated that contains high salt concentration, to extract any virus that may also not discharge from the fragment that pollutes.

Perhaps, allantoic fluid be can purify, liquid ingredient that purifies and the component that contains fragment formed.Preferred purifying method comprises centrifugal and filters.In high salt concentration solution, suspend or wash resulting precipitation or filter the back trapped substance with releasing virus, with described virus randomly merge to through or through in not centrifugal or the former allantoic fluid that alternate manner purifies.

Make processed supernatant liquor clarification generally can promote to be further purified by the sucrose density centrifugal.Typically use level to increase or the saccharose gradient of successive 30-50% (w/v).The manufacturers of influenza virus vaccine typically use the continuous flow centrifugation strategy.

The influenza virus alive of reclaiming from saccharose gradient at last, has the virus of significant proportion to be state of aggregation usually.Under the pH in the 6.5-8.5 scope usually, further handle separated gradient composition or component with the solution of the high level salt solution of phosphate buffered or other high ionic strength and merge thing (pool), make accumulative influenza virus release alive and make the virus yield maximization, these can be monitored by HA titre, analysis of infection, immunoassay and electron microscope.

That the back purification process of virus product has obtained depolymerization or the solution of monodispersed virion is ideally further handled this virion, and its loss is less than the virus loss that runs into usually.For example, usually not in full force and effect owing to have viral aggregate, the formalin deactivation of the virus of gradient purifying, and cause significant product loss and must carry out back formalin processing.Under the situation of influenza vaccines, after handling, formalin exist the live virus requirement to adopt ether extraction or other working method.After high salt was handled, the formalin of virus product was handled seldom failure, and has reduced the product loss that causes because of gathering widely.

The further advantage of the inventive method is that virus disintegrates down the purity that causes the viral raw material that reclaims and improves in allantoic fluid from fragment, i.e. the pollution that is caused by the egg component significantly reduces.Because the egg component as Protalbinic acid, can cause allergic reaction in some individualities, therefore method of the present invention is considered to the vaccine with higher degree can be provided, so the possibility that causes allergic reaction in accepting the individuality of vaccine descends.For example, one or more saccharose gradient components of handling the influenza virus that disintegrates down from the allantoic fluid fragment of the salt by high density generally are enough to provide detection not go out the product of Protalbinic acid.

Describe embodiment of the present invention with reference to following embodiment, these embodiment only are used for illustrative purposes and can not be used to limiting the scope of the invention.

Embodiment

Embodiment 1-is in by the allantoic fluid of influenza infection, and most of viruses exist with the form of highly insoluble virus/fragment aggregate

Passing through or do not pass through under the situation of centrifugal (Eppendorf Eppendorf centrifuge, 5000RPM, 5 minutes) purification, analyze thick allantois gleanings with HA (endpoint determination).

Table 1 shows that although record very high increment (influenza virus A/Texas), centrifugal purification typically makes the HA titre reduce by 4 times.Generally speaking, these data show that the most of influenza viruses that exist in the allantois gleanings are to exist with the low solubility form, are removed by physical method easily.

Embodiment 2The processing of-allantoic fluid has improved the titre of solvable influenza virus

The allantoic fluid that the thick influenza virus A/Moscow (InfluenzaA/Moscow) of 80 mul aliquots is infected mixes with 80 microlitre 3M NaCl solution.Viral sample and reference substance that salt was handled are cultivated 15 minutes on ice, and stir once in a while, purify by centrifugal (Eppendorf Eppendorf centrifuge, at full speed, 30 seconds) then.

HA analyzes: by with 50 microlitre sample transfer in the hole that contains 50 microlitre phosphate buffered saline buffers (PBS), with each supernatant samples (2 times) sequence serial dilution of 100 microlitres.Add the chicken RBC suspension of equal-volume (50 microlitre) 0.5% (v/v), mix, and at room temperature leave standstill (1-2 hour).In dilution sequence, measure the terminal point HA titre of each specimen as final hole to observe the complete agglutinative of hemocyte hole.

Typically, as shown in table 2, to compare with reference substance, as seen the HA titre has increased by 4 times in the allantoic fluid sample that infect in the influenza virus A that salt was handled/Moscow.

Embodiment 3The processing of-allantoic fluid fragment discharges soluble influenza virus

1.5M NaCl is applied to the gleanings of Flu A and B.Handle control sample with 0.15M NaCl, and with the centrifugal different number of times of each goods, to measure with respect to the viral sendout that is deposited in the supernatant liquor.

Each viral sample (2 * 300 microlitre) is carried out five equilibrium and mix with NaCl or the 0.15M NaCl (reference substance) of 1 volume 3M.With sample mix and be divided into 6 * 100 microlitres.After 30 minutes cultivate, under 10000RPM,, and collect 100 microlitre supernatant liquors and transfer on the HA analysis plates centrifugal 0,2,4 or 6 minute of sample (Eppendorf Eppendorf centrifuge).To precipitate and be suspended in again among the 100 microlitre 1.6M NaCl, centrifugal 2 minutes, and sedimentary washing lotion transferred on the HA assay plate.

The result is summarised in table 3 and 4.Numerical value is the HA terminal point of representing with HA unit/ml.

Table 3

Influenza virus B/sorb county (Yamanashi)

Table 4

Influenza virus A/Moscow (Moscow)

Embodiment 4-processing has greatly improved the virus yield through the influenza virus A of saccharose gradient purifying

The allantoic fluid sample that is used for high salt processing is cleaned by centrifugal, and washs 2 times with virus recovery from the fragment precipitation with the 1.6M salt washing (spending the night 1 hour then) of 10% volume.Purify washing lotion again, merge with the allantois supernatant liquor then.

Use raw glass fiber " degree of depth " filter cleaning control sample, with the typical method that uses in the simulation production of vaccine.After filtering, it is muddy slightly that control sample is.Do not use the strainer of 1 micron or 0.45 micron to carry out secondary filtration, therefore the scheme for worst based on the productive rate income of technology is provided.

On 6ml sucrose stagewise gradient, separate (fractionated) each allantois goods, obtain 17: 1 load-carry duty (loadingrate).With sample on the allantois sample on the gradient that contains several gradins, to obtain total load-carry duty.

Table 5

Influenza virus A/inferior gradient the application of sample of the many Buddhist nuns of new Galle

Experiment	The HAU that salt was handled	Contrast HAU	Reclaim ratio
				1	5232640	86816	60∶1
2	4149120	95488	44∶1

Table 6

Influenza virus A/Moscow gradient application of sample

Experiment	The HAU that salt was handled	Contrast HAU	Reclaim ratio
				1	368320	8944	40∶1
2	591200	9956	60∶1
				3	223232	7476	30∶1
4	289984	6516	44∶1

In all cases, when using the raw material of high salt processing, viral peak is very sharp-pointed, and is apparent that the peak of less and non-constant width when not carrying out this processing.Fig. 1 has provided under the situation of handling and not handling, the example of typical saccharose gradient curve.

Embodiment 5-Gao salt is handled the virus yield that has greatly improved through the influenza B of saccharose gradient purifying virus

Influenza B virus allantoic fluid sample is handled, and its processing mode is the same to influenza A virus treated mode with last embodiment.Use the raw glass fabric filter to purify control sample once more.On 6ml sucrose stagewise gradient, separate each allantois goods, to obtain 17: 1 load-carry duty.

Table 7

Influenza virus B/Hong Kong gradient application of sample

Experiment	The HAU that salt was handled	Contrast HAU	Reclaim ratio
				1	333312	123328	3∶1
2	780544	211968	4∶1
				3	727298	108032	7∶1

Embodiment 6-Gao salt is handled the titre that does not reduce virus infection

Use TCID ₅₀Mensuration process/do not have influenza goods, to estimate the influence of this processing to viral infection through the gradient purifying of too high salt processing.With the virus product five equilibrium, and each goods of equal portions are mixed with 1: 1 with 3M NaCl solution.Sample was cultivated on ice 1 hour, and centrifugal 5 minutes (Eppendorf Eppendorf centrifuge) purifies these samples under 6000RPM then.Serial dilution supernatant liquor and it being added on the mdck cell in the 96 hole assay plate in infecting medium.The CPE in each hole and/or HA state are used to estimate the existence of infection.Use the method (Amer.Jour.Hygiene, 27:493-497,1938) of Reed and Muench to calculate infection titer.

Table 8

Table 8 shows the titre that high salt is handled does not have negative impact live virus bacterial strain.Therefore, high salt is handled be used for allantois or other viral raw material can the break virus particle.

Embodiment 7-all relevant from the influenza data collection of HA analysis, infection titer and immunoassay acquisition

To after the saccharose gradient purifying, reclaim and by the HA assay determination component of influenza A/B gleanings of titre carry out optics immunoassay (OIA, Thermo BioStar).

Table 9

Number of components	The HA terminal point
		9	32768
10	131072
		11	524288
12	1048576

With the sample of PBS with each gradient composition of 1: 10,1: 100 and 1: 400 dilution, the aliquots containig with 100 microlitres is added in the BioStar sample hose that contains cracking agent then.Test kit explanation according to Biostar is measured, and the scale that provides in the contrast agents box is judged colour intensity rank (1-7).

Table 10

The erythrocyte agglutination analysis is virus/bacterial strain sensitivity, but all relevant to the ratio of red blood cell with virion.Just because of this, HA has reflected goods inner virus particulate quantity.The Flu OIA test of Thermo BioStar is a kind of tachysynthesis analysis that can report the existence of nucleoprotein influenza, therefore can infer the existence of virion.OIA colour intensity result is relevant with the HA titre of being measured.

Also to after the saccharose gradient purifying, reclaim and by the HA assay determination component of influenza A/B gleanings of titre carry out TCID ₅₀Analyze.

Table 11

The comparison of HA titre and infection titer in the gradient composition of selecting

Component	The HA titre	The HA ratio	The TCID titre	The TCID ratio
					5	256	1	1.94×10 5	1
14	524288	2048	3.73×10 7	192
					17	16384	64	1.50×10 7	77

Between analyzing, these have dependency, because the highest HA titre similarly has minimum infection titer corresponding to the highest infection titer and minimum HA titre.In order to help comparison,, HA titre ratio and TCID have been calculated with respect to measured Schwellenwert ₅₀The titre ratio.

Embodiment 8-processed being kept perfectly property of influenza virus

Carry out preliminary transmission electron microscopy (TEM) research, compared the peak value gradient composition of the influenza virus goods of influenza virus goods that salt handled and contrast.The copper TEM sample grid of Formvar-coating is swum on the drop (50 microlitre) of influenza virus A/new many Buddhist nuns of Galle Asia (New Caledonia) gradient composition, and adsorption sample 15 minutes at room temperature.With PBS washing grid 2 times, fix (5 minutes) with the PBS that contains 0.1% glutaraldehyde, use 0.2 micron filtering WFI water washing then 2 times and with 2% phospho-wolframic acid negative staining 1 minute.At air drying, on Hitachi H-7000 transmission electron microscope, use the acceleration voltage of 75kV to detect then in sample.Use Hamamatsu ORCAHR CCD photographic camera (AMT XR-60 image system), with the compression TIF form electron capture image of 12-bit gray scale.

Before gradient separations, in the goods of handling with high salt, observe, and virion seems complete form, and the same with untreated reference substance.Virosome has impervious complete coating of negative staining and outstanding surperficial furcella.All observed spherical and polymorphism virosome shape what handle in the two with the contrast goods.

The virosome of contrast in the goods usually combine with fragment and/or attached to the reticulin fiber pollutent on, as if this reticulin fiber pollutent concentrate or virosome sealed around virosome through negative staining.On the contrary, the virosome in the goods handled of salt mainly is monodispersed.In addition, in the goods that salt was handled, the character of the fibrous matrix of pollution seem to change and fibrous matrix and virosome between do not have tangible the combination.

Embodiment 9-gradient peak analysis revealed virus the density of being undertaken by refractometer is not changed because of processing

Analyzing the gradient composition of determining by HA is analyzed to measure the density corresponding to the HA peak activity by the light refraction instrument simultaneously.Use Misco PalmAbbe model PA200 to measure the refractive index of each gradient composition, the standard look-up table of use sucrose solution changes into density value again with refractive index.

Table 12: with the active corresponding specific refractory power of the saccharose gradient component peak value HA of influenza virus A/Texas allantois gleanings

Table 13: with the active corresponding specific refractory power of the peak value HA of the saccharose gradient component of influenza virus B/Hong Kong allantois gleanings

Summed up the refractive index of the saccharose gradient in influenza virus A/Texas and influenza virus B/Hong Kong in the table 12 and 13.For experimental period each time with for two kinds of test viruses, there is dependency closely in the HA peak value density of fraction of the allantois sample that salt is handled and the allantois sample of contrast.

Embodiment 10-dilution contains the allantoic fluid raising virus yield of virus before handling

Each allantois virus product (influenza virus B/mountain plough county and influenza virus A/Moscow) comprises that 4 samples (each 100ml) are used for the gradient purifying.Reference substance does not filter (sequence A) or passes through the purification (sequence B) of glass fibre degree of depth type filter.By adding the PBS of 0.5 volume, dilute processed sample in advance, making sample volume is 150ml, adds 20 * PBS of equal-volume (150ml) then, and cultivates 1 hour down to spending the night at 4 ℃.Use the hollow fiber filter of 500kda cutoff value, the sample reconcentration that these are processed is to 100ml with or without purifying (sequence C) or purifying (sequence D) through low-speed centrifugal.All samples carries out saccharose gradient purifying, separation and analyzes with HA measuring.

The result is summarised in table 14 and 15.

Table 14

Influenza virus B/mountain plough county

The group branch	Gradient [A]: filtering contrast allantoic fluid	Gradient [B]: contrast allantoic fluid	Gradient [C]: the allantoic fluid that salt was handled	Gradient [D]: salt is handled and filtered allantoic fluid
					1	5120	327680	20480	81920
2	10240	163840	163840	81920
					3	163840	163840	335544320	81920
4	655360	655360	5242880	2621440
					5	655360	327680	335544320	10485760
6	655360	163840	41943040	2621440
					7	655360	81920	327680	163840
8	327680	81920	40960	40960
					9	81920	40960	40960	20480
10	81920	20480	20480	20480
					11	40960	10240	10240	10240
12	40960	5120	10240	10240
					Total HA	3374080	2042880	718909440	16240640

Table 15

Influenza virus A/Moscow

The group branch	Gradient [A]: filtered contrast allantoic fluid	Gradient [B]: contrast allantoic fluid	Gradient [C]: the allantoic fluid that salt was handled	Gradient [D]: handle and filtering allantoic fluid through salt
					1	320	320	10240	10240
2	640	640	10240	20480
					3	1280	1280	20480	40960
4	2560	2560	81920	81920
					5	1280	2560	327680	81920
6	1280	1280	1310720	81920
					7	320	320	5242880	20480
8	20	160	2621440	5120
					9	80	160	40960	5120
10	40	160	20480	2560
					11	20	80	10240	2560
12	20	40	5120	2560
					Total HA	7860	9560	9702400	355840

No matter whether control sample passes through filtration, purification, and they have obtained testing with each the amount of viral essentially identical HA unit.Before gradient separations, do not have big precipitation through filtering reference substance.

On the contrary, obtain much higher HA titre through the goods that salt is handled than untreated reference substance then through dilution.Purification is not with virus branch band (banding) is necessary but purification can obtain the highest productive rate.The virus of removing by purification after salt is handled is not reclaimed best, and therefore with respect to the sample that does not purify, productive rate is lower.Yet, handle the remarkable improvement still realized with respect to the reference substance productive rate by dilution and salt in advance, and have nothing to do with purification before the gradient separations.Table 14 shows, for influenza virus B/mountain plough county, and with respect to filtering reference substance (sequence A), in not having the sample that purifies (sequence C), 213 times of gain in yield and in the sample that purifies (sequence D), 5 times of gain in yield.Table 15 shows, for influenza virus A/Moscow, and with respect to filtering reference substance, in not having the sample that purifies, 1234 times of gain in yield and in the sample that purifies, 45 times of gain in yield.

The foregoing example of practice it is evident that according to the present invention, by adjusted volume/salts contg, so that salt concn does not have height to making allantoic fluid protein precipitation (with the virus that is attached thereto), do not have low to the effect that can not play the virus of from the allantoic fluid fragment, dissociating best yet, can handle by using high salt concentration, make the viral largest optimization that from allantoic fluid, reclaims easily.Will carry out the allantoic fluid that is collected that salt is handled by initial analysis, and regulate the volume of the liquid that is collected, can easily carry out this optimization operation based on these initial tests.In such a way, can explain between batch of material and batch of material and proteinic variation in the allantoic fluid between possibility even bacterial strain and bacterial strain.

According to content disclosed by the invention, under the situation that does not have too much test, disclosed herein and claimed all compositions and/or method can be produced and implement.Although described composition of the present invention and method in the mode of preferred embodiment, but for skilled those skilled in the art, it is evident that, under the situation that does not break away from notion of the present invention, spirit and scope, can carry out various variations on the order to step in said composition and/or method and the described herein method or step.More particularly, it is evident that the alternative reagent described herein of some reagent that chemistry is all relevant with physiology two aspects is realized identical or similar result simultaneously.Conspicuous all this similarly the substituting with modification of skilled those skilled in the art are considered in defined spirit of the present invention, scope and notion by appended claim.Equally, the various bacterial strains of influenza virus A and B are grown in allantoic fluid and the improvement of the productive rate that reclaims from allantoic fluid although the above-mentioned embodiment that exemplifies all relates to, and method of the present invention is used in the allantoic fluid of the chicken embryo of virus infection typically other enveloped virus of growth easily.In fact, the rate of recovery of putting into practice relevant raising with the present invention may make that using the system of selection that becomes the virus of growing at present based on the viral growth of egg in mammalian cell cultures, condition is that virus is carried out the standard reorganization to adapt to this growth.

Reference cited herein is all introduced by reference particularly at this, and its degree is that the program that exemplifies or other details that they provide replenished those that enumerate herein.

Claims (38)

1. a method that reclaims virus from the allantoic fluid of the chicken embryo of virus infection contains fragment in the described allantoic fluid, and this method comprises the steps:

(a) one or more salt are joined make the total salt concentration that generates in it in the allantoic fluid greater than 0.5M; With

(b) reclaim the virus that from fragment, disintegrates down and be dissolved in the allantoic fluid.

2. method according to claim 1 wherein before allantoic fluid is separated from egg, joins salt in the allantoic fluid.

3. method according to claim 1, wherein recycling step (b) comprises that the allantoic fluid by centrifugal purification step (a) contains the precipitation of fragment with formation and contains viral supernatant liquor.

4. method according to claim 3 comprises that further by described precipitation being suspended in total salt concentration be virus is disintegrated down and to reclaim virus from resulting suspension from precipitation.

5. method according to claim 1, wherein recycling step (b) comprises that the allantoic fluid by filtration, purification step (a) contains the filtrate and the filtration trapped substance that contains fragment of virus with formation.

6. method according to claim 1, wherein to comprise that allantoic fluid with step a) carries out sucrose density centrifugal with location virus in density gradient for step (b).

7. method according to claim 1, wherein virus is enveloped virus.

8. method according to claim 7, wherein enveloped virus comprises rna gene.

9. method according to claim 8, wherein enveloped virus is to be selected from a kind of in orthomyxoviridae family, Paramyxoviridae, flaviviridae, batch Tectiviridae, Rhabdoviridae and the coronaviridae.

10. method according to claim 9, wherein virus is influenza virus.

11. method according to claim 10, wherein virus is influenza A virus.

12. method according to claim 10, wherein virus is influenza B virus.

13. method according to claim 1 is included in described one or more salt of adding and dilutes allantoic fluid before.

14. method according to claim 11, wherein virus is the Moscow bacterial strain of influenza A.

15. method according to claim 1, wherein the total salt concentration that generates in step (a) is 1.0M-3.5M.

16. method according to claim 15, wherein said one or more salt comprise sodium-chlor.

17. method according to claim 1, wherein said one or more salt are included in the solution of phosphate buffered.

18. according to the described method of claim 1, wherein with the pH regulator of allantoic fluid to or maintain in the scope of pH3-10.

19. from the allantoic fluid of the chicken embryo of virus infection, reclaim the method for virus, contain fragment in the described allantoic fluid, wherein allantoic fluid is purified, form liquid ingredient that purifies and the component that contains fragment, it is characterized in that, comprise from liquid ingredient that purifies and the component that contains fragment and extract virus, the described step of extracting virus from fragment comprises:

(a) be that the solution of 0.5M or bigger one or more salt will dissociate to the virus that the component that contains fragment combines in the suspension with total salt concentration; With

(b) from this suspension, reclaim by dissociated virus.

20. method according to claim 19, wherein said purification comprise that the centrifugal and described component that contains fragment comprises centrifugation.

21. method according to claim 19, wherein said purification comprise that filtration and the described component that contains fragment comprise the filtration trapped substance.

22. method according to claim 19 further comprises by purifying suspension forming second liquid that is cleaned and second component that contains fragment, thereby reclaim the step of virus from suspension.

23. method according to claim 22 further comprises by locating on sucrose density gradient, reclaims the step of virus from the liquid of second purification.

24. method according to claim 19, wherein virus is enveloped virus.

25. method according to claim 24, wherein enveloped virus comprises rna gene.

26. method according to claim 25, wherein enveloped virus is to be selected from a kind of in orthomyxoviridae family, Paramyxoviridae, flaviviridae, batch Tectiviridae, Rhabdoviridae and the coronaviridae.

27. method according to claim 26, wherein virus is influenza virus.

28. method according to claim 27, wherein virus is influenza A virus.

29. method according to claim 27, wherein virus is influenza B virus.

30. method according to claim 19 is included in described one or more salt of interpolation and dilutes allantoic fluid before.

31. method according to claim 28, wherein virus is the Moscow bacterial strain of influenza A.

32. method according to claim 19, wherein in described suspension, described total salt concentration is 1.0M-3.5M.

33. method according to claim 32, wherein said one or more salt comprise sodium-chlor.

34. method according to claim 19, wherein the pH of allantoic fluid is adjusted to or maintains in the scope of pH3-10.

35. reclaim the method for virus from the allantoic fluid of the chicken embryo of virus infection, wherein said virus is influenza virus, this method comprises the steps:

A) one or more salt are added in the allantoic fluid, generate 1.0 M or bigger total salt concentration within it the virus that combines with the component that contains fragment is dissociated in the allantoic fluid;

B), produce precipitation or trapped substance by centrifugal or filtration, purification allantoic fluid;

C) make the allantoic fluid of purification carry out the sucrose density gradient separation, location virus in density gradient; With

D) in gradient, separate the virus that is positioned.

36. method according to claim 35, wherein the pH of the allantoic fluid of step a) is adjusted to or maintains in the pH3.0-pH6.8 scope.

37. method according to claim 35, wherein the pH of the allantoic fluid of step a) is adjusted to or maintains in the pH6.8-pH9.8 scope.

38. method according to claim 35 further is included in the trapped substance that centrifugal precipitation that obtains among the salts solution inner suspension step b that 1.0M or bigger total salt concentration are provided or filtration obtain, and virus is separated from suspension.

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